L2M (Practical course) Script 2024 PDF

Master of Science in Biotechnology (MBT) Biotechnological Processes: from Lab to Market Laboratory Script Uzair Ahmed (M.Sc.) and Prof. Dr. Thomas Eisele  Contents Foreword 2 Day 1 — Introduction...

Master of Science in Biotechnology (MBT) Biotechnological Processes: from Lab to Market Laboratory Script Uzair Ahmed (M.Sc.) and Prof. Dr. Thomas Eisele  Contents Foreword 2 Day 1 — Introduction 3—15 Day 2 — Fast Protein Liquid Chromatography 16—20 Day 3 — Polyacrylamide Gel Electrophoresis 21—26 Day 4 — Enzyme kinetics 27—29 Lab report preparation – An important reminder 30—33 Appendix 1 34—35 Appendix 2 35 Appendix 3 36 Appendix 4 37 Appendix 5 38 Appendix 6 39 1 Foreword Dear Students, To work effectively in most life sciences’ laboratories, it is essential that you master key foundational skills, including the mathematical ‘rule of three’1 and the preparation of various reagents for biochemical experiments. Central to these tasks is the ability to perform accurate molar calculations and to safely pipette volumes as small as 10 μL — and even smaller. To ensure that all participants are at a comparable level of knowledge and skill, we will begin this practical course with a comprehensive review of essential laboratory techniques. The introductory sessions will cover the following topics: 1. Pipetting techniques 2. Basic calculations I - Variance and standard deviation 3. Basic calculations II – Dilutions 4. Calibration curves 5. Measurement of enzyme activity Proficiency in using 'Microsoft Excel' or an equivalent software program is critical for the success of your lab sessions. Simply generating data is insufficient; it is crucial that the data be analyzed and presented meaningfully and accurately. Each group is required to bring at least one laptop or tablet on lab day. Please ensure that all relevant Excel templates, such as those for calibration curves and enzyme activity measurements, are prepared in advance and ready to be used. 1 You are kindly requested to acquaint yourself with this concept before joining the laboratory. 2 Basic introduction – Day 1 (L2M) Introduction – Day 1 Achieving reliable results in the laboratory is contingent upon the use of precision instruments, such as (but not limited to) pipettes. Errors in pipetting can result from equipment malfunction or improper handling by the operator, leading to discrepancies in dispensed volumes that may compromise the accuracy and reproducibility of experimental outcomes. Therefore, it is imperative to regularly verify pipette calibration, typically every few months, to ensure accurate volume dispensation. Pipette calibration is a cornerstone of Good Laboratory Practice (GLP). It is essential that you develop proficiency in fundamental pipetting techniques. Pipetting tutorials from Eppendorf (viewing before lab is a prerequisite) How to pipette: https://www.youtube.com/watch?v=QGX490kuKjg Maintenance, disassembly, and assembly: https://www.youtube.com/watch?v=q0o-VBMVKio Pipette calibration: https://www.youtube.com/watch?v=fyOV9iyZMNY Basic calculations I – Variance and standard deviation Each group has to disassemble, service, and reassemble, at least, one pipette (under supervision) to become familiar with the parts that require maintenance Each group has to calibrate and prepare an excel sheet for the calibration of the following pipettes: 1 mL (100 μL – 1000 μL) pipette (blue color) 0.1 mL (10 μL – 100 μL) pipette (yellow color) Water (H2Odd) has a density of 1 g*mL-1 at 20°C and 1 ATM. This physical property of H2Odd allows the validation of dispensed volume of H2Odd by comparing it to the mass of the dispensed volume of H2Odd. The accuracy of a pipette can be determined by comparing the measured mass 3 Basic introduction – Day 1 (L2M) of dispensed volume with the theoretical mass of dispensed volume. This process is commonly known as ‘Gravimetric testing’. It is to be noted that changes in temperature and barometric pressure can influence the accuracy of measurements. In practice, the effect of pressure is negligible, however, potential effects from changes in temperature should be taken into consideration. The latter is accomplished by utilizing the ‘Z-factor’2, which is factored into the formula to calculate the volume of water that is dispensed: 𝐕𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐝𝐢𝐬𝐩𝐞𝐧𝐬𝐞𝐝 𝐇𝟐 𝐎𝐝𝐝 [𝐦𝐋] = Mass of dispensed H2 Odd [g] ∗ Z factor [mL ∗ g −1 ] Average calculated volume [mL] 𝐏𝐢𝐩𝐞𝐭𝐭𝐞 𝐚𝐜𝐜𝐮𝐫𝐚𝐜𝐲 [%] = ∗ 100 Theoretical volume [mL] For the blue pipette, please calibrate the following vol.: 100 μL, 500 μL, and 1 mL For the yellow pipette, please calibrate the following vol.: 25 μL, 50 μL, and 0.1 mL Note the temperature of water before beginning Hint: look at the temperature reading on the pH meter display For each calibration volume, conduct 10 volume transfers. Note the measured masses in your lab book. Calculate the volume dispensed and accuracy of the pipette using the above formulae3. Please also calculate the standard deviation and variance of your data using the following equations: Variance: Variance is a statistical measurement of the spread between values in a data set. Variance measures the distance of each number in the set from the mean, and thus from every other number in the set. Mathematically, variance is defined as the average of the squared differences from the mean and it is calculated as follows: 1 s2 = ∑ni=1(xi− X̅ )2 n−1 2 S = sample variance ∑ = sum of… 𝑥𝑖 = each value, 𝑋̅ = sample mean n = number of values in the data set 2 Z-factor depends on water temperature, air temperature, and pressure. ISO 8655 takes the air temperature to be equal to the water temperature. 3 Refer to appendix 2 for Z-factor values. 4 Basic introduction – Day 1 (L2M) Practice question: The calibration of an Eppendorf pipette was evaluated by individually weighing 10 volume transfers of 50 μL of H2Odd at 20°C. The results are given in table 1. Calculate the sample variance and standard deviation of the following data using your calculator and then using Microsoft Excel. Please also calculate the accuracy of this pipette. Table 1. Hypothetical data for the observed mass of 50 μL of H2Odd dispensed at 20°C. Transfer [-] Mass [mg] 1 46 2 58 3 52 4 44 5 43 6 62 7 64 8 50 9 52 10 60 Standard deviation: 𝒔 = +√𝒔𝟐 s = standard deviation s2 = sample variance Before moving on to the next task, please present your results to your supervisor and ask for their approval. 5 Basic introduction – Day 1 (L2M) Basic calculations II – Dilutions The process of dilution refers to the lowering of concentration. Why are dilutions important? Consider a scenario where 0.5 µg enzyme needs to be added a reaction. Based on the concentration of stock enzyme solution, it is determined that 0.5 µL of enzyme solution needs to be dispensed4. But what if a pipette capable of accurately dispensing such a small volume is not available? The solution is to dilute the stock enzyme solution. By performing a 10-fold dilution, 5 µL can be dispensed instead of 0.5 µL — while still delivering the required 0.5 µg enzyme. However, if the pipetting technique is not precise or the pipette is not properly calibrated, 4 µL might be dispensed instead of 5 µL — resulting in a 20% error. To further minimize this potential error, an additional 10-fold serial dilution (resulting in a 100-fold dilution overall)5 would allow the dispensation of 50 µL instead of 0.5 µL. Even if 48 µL is dispensed instead of 50 µL, the error would now be reduced to only 4%. Practice questions: 1. The total protein content of 2 mL Pichia pastoris supernatant is 0.5 µg. Please report the protein concentration as µg*μL-1. 2. What will be the resulting protein concentrations if the above supernatant is diluted 2- fold, 7.75-fold, 10-fold? Dilutions in general To understand dilutions, it can be helpful to consider the process of making juice from a fruit concentrate, such as orange juice. For instance, when preparing orange juice from a 100 mL concentrate, the instructions typically recommend adding 300 mL of water to the 100 mL of concentrate. This creates a 1:4 dilution, resulting in a mixture containing one-part fruit concentrate and three parts water, effectively making a 4-fold dilution. A key equation to remember when working with dilutions is: C1 ∗ V1 = C2 ∗ V2 In this equation: V1 represents the volume of the stock solution needed to achieve the desired dilution (this is often the unknown value to be calculated) C1 is the concentration of the stock solution 4 Practice: what is the concentration of stock enzyme solution in question? 5 Please refresh concepts about serial dilutions and multiplicative dilutions. 6 Basic introduction – Day 1 (L2M) V2 is the final or total volume of your required diluted solution C2 is the final concentration of your required diluted solution This equation is valid due to the 'Law of Conservation of Matter', which states that the amount of dissolved substance remains constant during the dilution process. In other words, the quantity of dissolved substance before dilution is equal to the quantity after dilution, ensuring that the equation balances. NOTE: Ensure that the units of volume and concentration are identical on either side of the equation. Molarity A mole can be defined in a number of ways. For instance, it can be a pigmented spot or mark on the human body; a burrowing insectivorous mammal; even a massive wall of stones laid in the ocean as a breakwater. For this course, however, a mole is the amount of substance that has a mass that is numerically equal to the molecular mass of the substance. Molarity (M) is a unit of concentration of a solution. It is the number of moles of a solute dissolved per liter of solution. A one molar solution contains one mole of solute per liter of solution. NOTE: “per liter of solution NOT solvent” → this distinction is very important! Unit conversion Attention to appropriate and consistent units is paramount! 1 mole = 103 millimole = 106 micromole 1 mmole = 1 millimole = 10-3 mole 1 µmole = 1 micromole = 10-6 mole = 10-3 millimole 1 nmole = 1 nanomole = 10-9 mole 1 g*l-1 = 1000 μg*mL-1 = 1000 mg*l-1 1% (w/v) = 10 g*l-1 Practice questions — Please solve for ‘X’: 1 mM = X mmole*l-1 = X mole*l-1 = X µmole*mL-1 1% (w/v) = X mg*mL-1 Practice questions — Calculate the following: 1. In a 50 mM solution of NaOH, how many grams of NaOH are present per mL? 2. How many μmole of a substance are present per mL in a 16 mM stock solution? 7 Basic introduction – Day 1 (L2M) 3. You have just plotted a standard curve for a protein concentration determination. One of the standards had 24 μg protein in a volume of 60 μL. Express the concentration of this standard as mg*l-1. 4. A volume of 0.1 mL of a 1:10 dilution of an unknown sample contains 320 μg of protein. What is the concentration of the undiluted sample in mg*mL-1? 5. You have a stock solution of 0.8 M Tris-HCl buffer. You need to make 250 mL of Tris- HCl with a final concentration of 10 mM. How will you formulate this solution? 6. The protocol for an enzyme assay requires that you add 20 μL of a 100 mM solution of p-nitrophenol phosphate to a total volume of 3.03 mL (this final volume includes the 20 μL that you added). What is the final concentration of the p-nitrophenol phosphate? 8 Basic introduction – Day 1 (L2M) Protein quantification and enzyme activity determination The evaluation of formed product(s) or depleted substrate(s) inside a shake-flask or a bioreactor is a pivotal part of a successful fermentation. Industrially, Trichoderma reesei is most commonly cultivated filamentous fungus for the production of endo- and exo-cellulases as well as β-glucosidases. Hence, the determination of these enzymes’ activities and their quantification is crucial for the optimization of bioreactor fermentations or any other scale of fermentation. Estimating the protein content of an enzyme solution is a prerequisite to determine specific enzyme activity. Each group has to determine the protein content of a commercial Trichoderma reesei product, Methaplus® (DSM, Netherlands), after appropriate dilution(s) Also, each group has to determine the cellobiohydrolase I (CBH-I) activity present inside Methaplus® (DSM, Netherlands) Determination of protein concentration according to the method of Marion Bradford To estimate the protein concentration of a given sample, the Roti®Nanoquant6 assay (5X, Carl Roth, Germany) will be used. This is a colorimetric assay based on the method first established by Bradford (1976). The dye contained in the staining solution is Coomassie Brillant Blue G- 250 (CBBG). CBBG belongs to the Coomassie family of triphenylmethane dyes. Under acidic conditions, proteins bind to the Coomassie dye. This results in a spectral shift from the reddish- brown form of the dye to the blue form monitored at wavelengths of 450 nm and 590 nm (Figure 1). The increase in absorption is proportional to the protein concentration in the sample. The calibration will be carried out using bovine serum albumin (BSA; Biowest, France) as the standard protein. Figure 1. Three states of CBBG 250 at different wavelengths. Two calibration curves with BSA (in H2Odd) have to be prepared for the following concentration ranges (prepare, at least, 500 μL of each standard): 6 Additional information can be found in appendix 1. 9 Basic introduction – Day 1 (L2M) Calibration range 1: 0 μg*mL-1 — 150 μg*mL-1 Calibration range 2: 0 g*L-1 — 1 g*L-1 NOTE: 0 μg*mL-1 and 0 g*l-1 = blank | Label your standards appropriately and have them stored at -20°C before leaving the lab. You will need to reuse these standards during the next labs to prepare fresh calibration curves. For protein quantification with Roti®Nanoquant (1X)7 in 96-well plate format, the pipetting scheme is detailed in table 2. The unknown protein concentration of samples is calculated by [−] 590nm using the equation generated by plotting the blank-corrected quotient, [−] 450 nm , of known BSA standards against their concentrations. Table 2. Pipetting scheme for protein quantification via the Bradford assay using calibration range 18: 0 μg*mL-1 — 150 μg*mL-1. Reagent Volume [μL] BSA standards and/or unknown sample 50 Roti®Nanoquant (1X) 200 Incubate for 5 minutes at room temperature & measure the absorbance at 450 nm & 590 nm Table 3. Pipetting scheme for protein quantification via the Bradford assay using calibration range 29: 0 g*L-1 — 1 g*L-1. Reagent Volume [μL] BSA standards and/or unknown sample 15 Roti®Nanoquant (1X) 200 Incubate for 5 minutes at room temperature & measure the absorbance at 450 nm & 590 nm 7 Diluted from the 5X stock solution. 8 All analyses have to be carried out, at least, as duplicate measurements. Avoid forming air bubbles. In case of excessive bubbling, use flame (via a lighter) to disrupt the bubbles. 9 All analyses have to be carried out, at least, as duplicate measurements. Avoid forming air bubbles. In case of excessive bubbling, use flame (via a lighter) to disrupt the bubbles. 10 Basic introduction – Day 1 (L2M) Cellobiohydrolase I (CBH-I) assay Background Cellobiohydrolase (CBH) is an enzyme that plays a crucial role in the breakdown of cellulose, a major component of plant cell walls. Specifically, CBH is a type of cellulase enzyme that catalyzes the hydrolysis of cellulose by cleaving off cellobiose units (a disaccharide composed of two glucose molecules) from the ends of the cellulose chains. There are two main types of CBHs: 1. Exo-cellobiohydrolase (CBH-I; EC 3.2.1.76): This type attacks the reducing ends of cellulose chains. 2. Exo-cellobiohydrolase (CBH-II; EC 3.2.1.91): This type attacks the non-reducing ends of cellulose chains. Together with other cellulases, such as endoglucanases and beta-glucosidases, CBHs contribute to the complete hydrolysis of cellulose into glucose. This process is essential in nature for the decomposition of plant material and is also important in various industrial applications, such as the production of biofuels, where cellulose is converted into fermentable sugars Principle CBH-I acts predominantly on the reducing ends of cellulose chains. It cleaves off cellobiose units (two glucose molecules linked by a β-1,4 bond) from the reducing end. CBH-I activity can be measured via a reporter reaction using a synthetic substrate. This reaction involves the release of a chromophore, namely, 4-nitrophenol (pNP), that can be measured spectrophotometrically. 4-nitrophenyl-β-D-lactopyranoside (pNPL)10 is a disaccharide derivative of β-lactose in which the anomeric hydroxy hydrogen is replaced by a 4-nitrophenol group. CBH-I cleaves the bond between nitrophenol and glucose. The liberated pNP can then be detected at 405 nm. Safety Wear laboratory goggles, lab coat, and gloves (when necessary). The chemicals used in this assay are identified by the following H-phrases (hazard). All the recommendations elaborated in the P-phrases (precautionary) are to be followed (table 3). 10 pNPL is not the only synthetic substrate used for detecting CBH-I activity 11 Basic introduction – Day 1 (L2M) Table 4. P- and H-phrases for the CBH-I assay to be considered for your personal safety11. Substance Warning symbols H-phrases P-phrases Sodium carbonate H319 P305+P351+P338 P261, H301, M312+H332, P301+P310+P330, 4-nitrophenol H373 P302+P352+P312, P304+P340+P312 P210-P260-P280-P303 + P361 + P353-P305 + Acetic acid H226-H314 P351 + P338-P370 + P378 4-nitrophenyl ß-D- H302-H371 P260 lactopyranoside Methaplus H334 P261 - P284 - P501 Devices and consumables Thermoshaker Pipettes & pipette tips 1.5 mL & 2 mL microfuge tubes 96 well flat-bottom microtiter plates12 Microtiter plate reader Centrifuge pH meter Chemicals Sodium acetate (molecular mass: 82.03 g*mol-1) 11 Descriptions of the phrases can be found in the Moodle course under ‘Lab Safety Instructions’. 12 Please do not touch the bottom of the plates. Hold the plates at the sides (perimeter). 12 Basic introduction – Day 1 (L2M) Acetic acid (liquid) ≥99% Sodium carbonate (molecular mass: 105.99 g*mol-1) p-nitrophenol (pNP; molecular mass: 139.11 g*mol-1) 4-nitrophenyl β-D-lactopyranoside (pNPL; molecular mass: 463.4 g*mol-1) NOTE: it is your task to learn about the substrate’s solubility and the best way to make a solution at the required concentration. Buffers Buffer for dissolving and/or diluting enzyme, substrate, and pNP: 50 mM sodium acetate buffer pH 5.0 1 M sodium carbonate (stop solution) Calibration, linearity, and accuracy pNP liberation by CBH-I is estimated by using a pNP calibration curve within the range of 0 mM—2 mM. The valid measuring range of absorbance values at 405 nm for enzyme activity determination after blank-correction is between 0.05—1.0. Pipetting scheme for the preparation of para-nitrophenol calibration curve is shown in table 413. Table 4. Pipetting scheme for the preparation of pNP calibration curve14. in Sample in Blank Reagent [μL] [μL] Buffer 100 125 At least 5 different pNP concentrations 25 0 (0 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM) 1 M Na2CO3 100 100 Mix well — either by pipetting or by vortexing. Transfer 150 μL in a microtiter plate well and measure the absorbance at 405 nm NOTE: 0 mM pNP = blank = water | Label your standards and substrate solution appropriately. Please have them stored at -20°C before leaving. You will need to reuse these standards during the next labs to prepare a fresh calibration curve. 13 It is to be noted that the volumetric ratios of all species are identical to the ratios used in table 5. 14 All analyses have to be carried out, at least, as duplicate measurements. 13 Basic introduction – Day 1 (L2M) Enzyme sample(s) should be clarified by centrifugation (30 seconds) before measurement. Enzyme sample(s) should be diluted, at least, 50-fold. In case of doubt, e.g. in case of very heavily buffered starting solutions or low dilutions, the pH of the respective dilution must be checked. Enzyme dilutions should be selected based on the protein concentration of the starting solution. If no data/empirical values are available, the following dilutions should be tested: 1:10, 1:50, 1:100, 1:1000. The pipetting scheme for the determination of the CBH-I activity is shown in table 5. Table 5. Pipetting scheme for CBH-I assay7. in Sample in Blank Reagent [μL] [μL] Buffer 50 50 Diluted enzyme 50 0 Incubation15 at 50°C for 2 minutes 2 mM pNPL 25 25 Incubation16 at 50°C for 5—3017 minutes 1 M Na2CO3 100 100 Diluted enzyme 0 50 Transfer 150 μL in a microtiter plate well and measure the absorbance at 405 nm18 𝐄𝐧𝐳𝐲𝐦𝐞 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = mkat ∗ l−1 concentration of 𝑝NP [mM] total reaction volume [mL] 𝐄𝐧𝐳𝐲𝐦𝐞 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = ∗ ∗ DF reaction time [sec] volume of enzyme added [mL] where; DF = dilution factor of the starting enzyme solution total reaction volume [mL] = dilution factor of the enzyme inside the reaction volume of enzyme added [mL] 15 This incubation step is performed to ensure that the system is equilibrated at the desired temperature 16 This incubation step is performed to hydrolyze the substrate with enzyme 17 The duration of reaction is a variable parameter dependent on substrate concentration and enzyme dilution 18 This reading has to be compared to the pNP calibration curve to estimate the amount of pNP liberated 14 Basic introduction – Day 1 (L2M) Explanation for enzyme activity calculation: According to the SI system, Katal (kat) is the amount of enzyme that converts 1 mol substrate per second under specified conditions. Consider the following example, a 10 mL solution of a 2-fold diluted enzyme is being used. A volume of 50 μL of this diluted enzyme is added in a total reaction volume of 225 μL (50 μL+ 175 μL). The reaction is stopped after 3 minutes. Based on the pNP calibration curve, it is estimated that that this enzymatic reaction liberated 2 mM pNP. concentration of 𝑝NP [mM] total reaction volume [mL] 𝐄𝐧𝐳𝐲𝐦𝐞 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = ∗ ∗ DF reaction time [sec] volume of enzyme added [mL] 2 mM 0.225 mL 𝐄𝐧𝐳𝐲𝐦𝐞 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = ∗ ∗ 2 = 0.1 mkat ∗ L−1 180 sec ∗ L 0.05 mL 1 liter of undiluted enzyme solution contains 0.1 mkat enzyme activity (0.1 mkat*L-1) 1 mL of the undiluted enzyme contains 0.1 μkatal enzyme activity (0.1 μkat*mL-1) 1 liter of diluted enzyme solution contains 0.05 mkat enzyme activity (0.05 mkat*L-1) 10 mL of the diluted enzyme solution contains 0.5 μkat enzyme activity 15 Fast Protein Liquid Chromatography (FPLC) – Day 2 (L2M) Fast Protein Liquid Chromatography (FPLC) – Day 2 The purification of (un)known proteins is a frequent task in biochemistry labs. Please make yourself familiar with the underlying principles behind purification of proteins (e.g. FPLC lecture). Briefly, ion-exchange chromatography (IEX) separates molecules on the basis of differences in their net surface charge. Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density, and surface charge distribution. IEX takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific protein. In an IEX separation, reversible interactions between charged molecules and oppositely charged IEX media are controlled in order to favor binding or elution of specific molecules and achieve separation. A protein that has no net charge at a pH equivalent to its isoelectric point (pI) will not interact with a charged medium. However, at a pH above its pI, a protein will bind to a positively charged medium or anion exchanger and, at a pH below its pI, a protein will bind to a negatively charged medium or cation exchanger (Figure 2). In addition to the ion-exchange interaction, other types of binding can occur, but these effects are very small and mainly due to van der Waals forces and nonpolar interactions. Figure 219. Net charge of a protein as a function of pH, showing the pH ranges in which protein is bound to anion or cation exchangers. The pH range over which the protein is stable may be only a small fraction of the binding range; this must also be taken into consideration when choosing an ion-exchange medium. 19 Alan Williams and Verna Frasca, Current Protocols in Protein Science (1999) 8.2.1-8.2.30. 16 Fast Protein Liquid Chromatography (FPLC) – Day 2 (L2M) IEX [usually] consists of 5 phases: equilibration, sample application, column wash, elution, regeneration, and re-equilibration. A representative chromatogram of an IEX is depicted in figure 3. Figure 3. Chromatogram of a typical IEX separation (Ahmed et al. 2022) using linear gradient elution. The UV (black) and conductivity (grey) traces show the elution of protein peaks and the changes in salt concentration during elution. Increase in conductivity indicates increase in salt concentration. Each group has to purify CBH-I from Methaplus® via anion-exchange chromatography (AIEX)20. Each group also has to prepare a purification summary table. 20 Why AIEX? You’re expected to answer this question on the day of the lab. 17 Fast Protein Liquid Chromatography (FPLC) – Day 2 (L2M) AIEX purification of CBH-I from Methaplus® Column: HiTrap Q FF 1 mL (Sepharose fast flow: 6% highly cross-linked beaded agarose matrix; strong anion exchanger) Dynamic binding capacity of the column: ~70 mg Ribonuclease A per mL of resin Pressure limit of the column: 0.5 MPa Binding buffer (A): 20 mM Tris-HCl, pH 7.2 (500 mL) Elution buffer (B): 20 mM Tris-HCl+ 1 M NaCl, pH 7.2 (500 mL) FPLC equipment: ÄKTA™ Go and ÄKTA™ Start Control software: UNICORN™ version 7.x Preparatory steps: 1. Set the pressure limit of the column in the control software. 2. Flush out the storage solution (20% ethanol) from the column using either H2Odd or binding buffer at a flow rate of 4 mL*min-1. NOTE: Do not use elution buffer as the high salt content might precipitate upon contact with ethanol. 3. Manually equilibrate the FPLC system on the starting (sample binding) conditions at a flow rate of 4 mL*min-1. 4. Program a purification method using the control software. 5. Dilute Methaplus® 80-fold into a final volume of 15 mL binding buffer in a suitable vessel. 6. Filter the diluted sample using a syringe fitted with a 0.22 μm or 0.45 μm filter into a measuring cylinder. 7. Put clean 15 mL tubes in the fraction collector. Anion-exchange (IEX) method (approximate run time ≤20 minutes): 1. Load 10 mL of the filtered sample onto the column using the sample pump at a flowrate of 4 mL*min-1. Collect the flow-through in 10 mL fractions. Monitor protein elution at 280 nm. NOTE: save the remaining ~5 mL sample for protein quantification, enzyme activity test, and PAGE. 18 Fast Protein Liquid Chromatography (FPLC) – Day 2 (L2M) 2. ‘Wash’ the column with 40 column volumes (CV) of binding buffer (A) at a flowrate of 4 mL*min-1 to remove unbound protein. Collect the flow-through in 10 mL fractions. Monitor protein elution at 280 nm. 3. Start the elution phase using a linear gradient of 0%—100% elution buffer (B) in 20 CV at a flowrate of 4 mL*min-1. Collect 2 mL fractions (fixed volume fractionation). 4. Continue the elution phase at 100% slution buffer (B) for 5 CV. Collect 2 mL fractions (fixed volume fractionation). 5. Regenerate the column with 10 CV of elution buffer at a flowrate of 4 mL*min-1. Collection of flow-through at this stage is not needed. 6. Re-equilibrate the column with 20 CV of binding buffer at a flowrate of 4 mL*min-1. Collection of flow-through at this stage is not needed. The following tasks have to be performed: 1. Preparation of purification buffers, enzyme sample, collection tubes. 2. Preparation of BSA calibration curve to determine protein concentration of the enzyme and of the samples from the FPLC. 3. Programming a purification method using the control software. 4. Running the purification method. 5. Determination of enzyme activity in the applied sample and collected fractions21. Determination of protein concentration in the applied sample and in the FPLC samples in which CBH-I activity is detected. NOTE: prepare new BSA and pNP calibration curves for the day using previously prepared standards. 6. Pooling the fractions containing CBH-I activity, determining the pool’s protein content and total enzyme activity. 7. Storing the applied sample (crude enzyme) and ‘purified’ active CBH-I at -20°C in appropriately labelled tubes for later experiments (SDS-PAGE and enzyme kinetics). 8. Preparation of a protein purification table. Protein purification table A protein purification table should allow a reader to easily evaluate the procedure and readily detect particularly effective and ineffective purification steps. It should be easy to see if large losses occurred at a particular step. A suitable table will contain the following columns: major 21 All analyses have to be carried out, at least, as duplicate measurements. 19 Fast Protein Liquid Chromatography (FPLC) – Day 2 (L2M) steps in purification, amount of total protein, amount of target protein activity or total activity, specific activity, yield, purity, and/or purification fold. Table 6 depicts an example of protein purification able. Table 6. A typical protein purification summary table. 20  Polyacrylamide gel electrophoresis – Day 3 (L2M) Polyacrylamide gel electrophoresis – Day 3 Table 7. P- and H-phrases for to be considered for your personal safety22. Substance Warning symbols H-phrases P-phrases P201 P280 P301+P312 H319H302-H315-H317- Acrylamide P302+P352 H319-H340-H350- solution P305+P351+P338 H361f-H372 P308+P313 P210 P280 H225-H302+H332- P301+P330+P331 TEMED H314 P303+P361+P353 P305+P351+P338 P310 P261 P280 P302+P352 H272-H302-H315- APS P305+P351+P338 H317-H319-H334-H335 P332+P313 P337+P313 H301+H331-H310- P273 P280 P302+P352 2- H315-H317-H318- P304+P340 Mercaptoethanol H373-H410 P305+P351+P338 P310 P210 P261 P280 Sodium dodecyl H228-H302+H332- P302+P352 sulfate H315-H318-H335-H412 P305+P351+P338 P312 The preparation of polyacrylamide gels will be carried out according to the method of Laemmli (1970). Various acrylamide concentrations can be used to match the mobility of the analyzed proteins. Equipment used for electrophoresis in this experiment is called ‘Mini Protean 3’, manufactured by Bio-Rad. Mini Protean 3 consists of: casting stand, casting frame, clamping frame, silicone rubber gaskets, well combs, electrode assembly, inner chamber, outer chamber, glass short plates, and glass spacer plates (bonded gel spacers that determine the thickness of the gel). 22 Descriptions of the phrases can be found in the Moodle course under ‘Lab Safety Instructions’. 21 Polyacrylamide gel electrophoresis – Day 3 (L2M) Figure 2. Overview of Mini Protean 3 system components (from Mini Protean® 3 Instruction Manual 165-3301/165-3302). Hand-casting gels for electrophoresis Wearing gloves and ensuring proper usage is mandatory while handling electrophoresis equipment and preparing gels. The glass plates, well combs, casting frame, and casting stand have to be cleaned well. The glass plates can be cleaned with 80% ethanol and lint-free tissue paper. The casting frame, casting stand, and well combs can be cleaned with H2Odd and lint-free tissue paper. The gel cassette can be assembled by following the pictorial depiction in figure 4. Ensure that all the components are placed flush against a flat surface at the time of assembly. Figure 4. Preparation of the gel cassette assembly (from Mini Protean® 3 Instruction Manual 165-3301/165-3302). 22 Polyacrylamide gel electrophoresis – Day 3 (L2M) Once the gel cassette has been assembled, please check for leaks. Do not undo the assembly once a leak-free system has been validated! Place a comb into the assembled gel cassette. Mark the glass plate 1 cm below the comb teeth. This is the level to which the resolving gel has to be poured (~80% height of short plate). You can also choose to mark the wells at this stage. Remove the comb. Now, you can begin prepare the gel solutions. Gel preparation, gel casting, and sample preparation has to be carried out under a fume hood. The gel compositions for the preparation of two SDS-PAGE gels (1.5 mm) and two Native- PAGE gels (1.5 mm) are shown in table 8. Stock solution of acrylamide is referred to as ‘matrix’. All chemicals except tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) have to be mixed gently in 15 mL or 50 mL tubes. Avoid formation of bubbles. As soon as TEMED (a polymerization catalyzer) and APS (the radical polymerization starter) are added, the resolving gel solution has to be gently mixed by inversion and subsequently transferred into the space between the two glass plates (~80% of total height of the short plate = ~6 mL for a 1.5 mm gel). Polymerization takes a minimum time of 35 minutes. To avoid evaporation, the gel has to be overlaid with isopropanol. The layer of isopropanol also straightens the top of resolving gel. The stacking gel solution has to be prepared analogously to the resolving gel solution (table 8). Isopropanol has to be removed completely before the stacking gel can be poured — this can be achieved by washing it once with H2Odd and then drying. The stacking gel solution will be transferred into the space between the two glass plates (~3 mL for a 1.5 mm thick gel) until it overflows slightly. The comb has to be inserted between the plates carefully and immediately. The maximum loading volume for one well of a 10 well comb (1.5 mm) is ~66 μL. After ~35 min of polymerization, the comb can be removed (optional at this stage) and the gel can be taken out of the casting frame. PAGE gels can be stored at 4°C for up to one week if they are wrapped in a wet paper towel and sealed in an airtight container. 23 Polyacrylamide gel electrophoresis – Day 3 (L2M) Table 8: Composition of acrylamide gels (resolving and stacking; 2 of each) for SDS-PAGE. Use H2Odd instead of SDS in the recipe to prepare Native-PAGE gels. Resolving gel Stacking gel 8 4 concentration [%] concentration [%] Matrix concentration Matrix concentration 30 30 [%] [%] Total volume [mL] 15 Total volume [mL] 10 Resolving Gel Stacking Gel Components Volume [μL] Components Volume [μL] 1.5 M Tris pH 8.8 1250 0.5 M Tris pH 6.8 1250 H2Odd 9485 H2Odd 7232 (7230) 10% (w/v) SDS23 150 10% (w/v) SDS12 100 Matrix (37.5:1) 4000 Matrix (37.5:1) 1333 (1330) TEMED 15 TEMED 10 10% (w/v) APS12 100 10% (w/v) APS12 75 Total Volume 15000 Total Volume 10000 Sample preparation for [reducing] SDS-PAGE and Native-PAGE Protein samples are usually standardized between 2 μg—5 μg total protein content per well for PAGE analysis (see appendix 5 for an example calculation). For SDS-PAGE, after being thoroughly mixed [via vortex] with the reducing sample loading buffer (SLB)24, samples have to be centrifuged and then heated at 95°C for 5 minutes. Afterwards, the samples have to be briefly cooled on ice and centrifuged once more (15 sec, table centrifuge). Subsequently, samples have to be applied onto the gel. For Native-PAGE, samples have to be thoroughly mixed [via vortex] with the native sample loading buffer (SLB)13 and centrifuged (15 sec, table centrifuge). Subsequently, samples have to be applied onto the gel. Unstained and prestained protein markers have to loaded on to the SDS-PAGE gel alongside samples. Any empty well has to be loaded with 1X SLB 23 APS and SDS solutions (1 mL each) have to be freshly prepared right before casting the gels. 24 Sample loading buffers (SLBs) will be provided by the supervisors. 24 Polyacrylamide gel electrophoresis – Day 3 (L2M) The composition of representative sample loading buffers for PAGE under reducing, non- reducing, and native conditions is shown in table 9. Table 9. Compositions of representative sample loading buffers (1X) for electrophoresis under various conditions. Concentration Chemical Reducing SDS- Non-reducing SDS- Native-PAGE PAGE PAGE Tris-HCl 50 mM 50 mM 50 mM SDS 2% (w/v) 2% (w/v) 0% (w/v) Glycerol 10% (w/v) 10% (w/v) 10% (w/v) Bromophenol blue 0.02% (w/v) 0.02% (w/v) 0.02% (w/v) 2-mercaptoethanol* 5% (v/v) 0% (v/v) 0% (v/v) *some recipes use DTT instead of 2-mercaptoethanol Electrophoresis and subsequent staining of the polyacrylamide gels Samples and marker proteins (pre-stained and un-stained; 3 μL—10 μL) have to be transferred into separate wells. A constant voltage of 120 volts is applied (Powerpack System 300, Bio- Rad Laboratories GmbH, Munich). At the end of electrophoresis, the gels have to be removed from the glass plates and then stained. The composition of running buffers (1X) for PAGE analyses is shown in table 10. Table 10. Running buffer (1X) composition for SDS-PAGE and Native-PAGE Concentration [g*l-1] Chemical SDS-PAGE Native-PAGE Tris base 3.03 3.03 Glycine 14.44 14.44 SDS 1.0 0 Coomassie staining PAGE gels and subsequent digitization A ready-to-use staining solution and the staining protocol will be provided by the supervisors. Stained gels will be digitized using the GelDoc Go Gel Imaging System (Bio-Rad Laboratories GmbH, Munich). 25 Polyacrylamide gel electrophoresis – Day 3 (L2M) Molecular weight estimation of unknown proteins via SDS-PAGE Please refer to appendix 6 for detailed instructions. It is advised that the image(s) of the gel(s) be printed to simplify the determination of migration distances. 26 Enzyme kinetics and biochemical characterization – Day 4 (L2M) Enzyme kinetics and biochemical characterization – Day 4 Enzyme-catalyzed reactions are model concepts to understand the conversion of substrates into products with the help of isolated enzymes serving as catalytically active substances. A distinction is made between one-substrate reactions [considering the activating substances and the reaction conditions] and the more complex multi-substrate reactions. The Michaelis-Menten equation is the mathematical formulation for the model "enzyme- catalyzed reaction with a substrate component". The equation connects the initial velocity ‘V0’ and the initial substrate concentration ‘S’ with the constant ‘Km’. The initial rate of product formation is determined at different substrate concentrations, constant enzyme activities and operating conditions. The graphical evaluation by means of Hanes–Woolf linearization or by non-linear regression deploying a software tool leads to Km and Vmax values. The theoretical basics will be thought in the lecture Biotechnological processes: from Lab to Market. Determination of kinetic parameters of the purified -Glucosidase (BGL) Each group will determine the kinetic parameters of the purified CBH-I. Different concentrations of substrate will be used to determine the initial velocity of the purified CBH-I. Five different concentrations of substrate solution have to be prepared, as well as an inhibitor stock solution in 50 mM sodium acetate (pH 5.0). Substrate concentrations: 1 mM, 2.5 mM, 5 mM, 7.5 mM, and 10 mM (in 50 mM sodium acetate buffer, pH 5.0). Glucose inhibitor solution: 0 mM, 50 mM, and 100 mM (in 50 mM sodium acetate buffer, pH 5.0). In order to stay within the linear range of the substrate conversion or product formation, it´s important that the Absorbance405 nm stays within the range of 0.1—1.0. If your absorbance measurements exceed the aforementioned range, please make sure that you redo the analysis or dilute your sample before measurement. An exemplarily pipetting scheme is depicted in table 11 for conducting the reactions. 27 Enzyme kinetics and biochemical characterization – Day 4 (L2M) Table 11. Exemplary pipetting scheme for the determination of the kinetic parameters of the purified BGL25. in Sample in Blank Reagent [μL] [μL] Buffer 33 33 Inhibitor solution 33 33 Diluted enzyme 33 33 Incubation at 50°C for 5 minutes Substrate 25 25 Incubation at 50°C for 5—30 minutes 1 M Na2CO3 100 100 Diluted enzyme 0 33 Transfer 150 μL in a microtiter plate well and measure the absorbance at 405 nm Parallel to the measurement of the enzyme activity (5 different substrate concentrations), the linearization according to Hanes-Wolf, Lineweaver-Burk, Eadie-Hofstee, and the application according to Michael-Menten will be carried out. Furthermore, a non-linear regression has to be performed using Microsoft Excel. If individual measuring points show deviations, please redo the measurements. Partial characterization of the purified CBH-I (optional) Experiments to biochemically characterize the purified BGL will be conducted. Temperature maximum As the reaction temperature rises, the Gibbs energy of the system and thus the reaction speed increase. However, as the temperature continues to rise, a point is reached at which the supplied energy is sufficient to break hydrogen bonds and other bonds that are involved in maintaining the tertiary structure of the enzyme so the enzyme loses its activity. The point at which the increase in the reaction rate with the temperature is compensated for by the decrease in the reaction rate as a result of the destruction of the tertiary structure is referred to as the temperature 25 All analyses have to be carried out, at least, as duplicate measurements. Moreover, please analyze a blank for each substrate concentration. 28 Enzyme kinetics and biochemical characterization – Day 4 (L2M) maximum. The effect of the temperature on the initial activity of the purified BGL determined in a range of 20°C—70°C with a step size of 10°C using the standard activity buffer and pipetting scheme. pH optimum An enzyme is only catalytically active within a certain pH range. An enzyme has its highest activity at a specific pH value, which is known as the pH optimum. At pH values that are relatively far from the pH optimum, the enzyme begins to denature and the conformation required for catalytic activity is disturbed. The pH optimum of the purified BGL is determined in the pH range from 3 to 6 in steps of one. Sodium citrate buffer (pH 3.0 – 6.0) and sodium acetate buffer (pH 3.6 – 5.5) are used. Two different buffer systems are being applied in order to test for so-called buffer effect. Experimental data indicates that buffer salts tend to affect an enzyme’s activity. The optimum pH is determined using the standard. 29 Lab report preparation (L2M) Lab report preparation – An important reminder Your results and lab reports will account for 50% of the total grade of the practical course, Biotechnological Processes: from Lab to Market. Properly formatted26 lab report for each day has to be submitted27 in PDF file format via email28 no later than 2 weeks after the lab. !!! PDF files are to be named in this format: G1.1, Experiment 1, 2024 Lab report files not named like this will not be considered. First submissions will be considered final. You are encouraged to get your questions answered and doubts clarified before submission via email16 or in-person29. The independently prepared laboratory reports by each group must contain the following points: 1. An independently written introduction encompassing the rationale behind all the conducted experiments. The introduction should also contain, at least, 5 scientific literature references30. 2. Copy-pasting material and methods part from the provided script is forbidden. Only mention differences to the prescribed methodology that were improvised in the lab. Plagiarizing material from existing scientific literature will result in a failing grade for the lab report. 3. In the results section, please use tables and figures13 to elaborate the obtained data. Ensure consistency with unit usage. Attach raw data ONLY when necessary. 4. Tables need to be titled with a self-explanatory heading and figures need to be provided a self-explanatory caption (examples can be found throughout this script)13. 5. Please make sure that you refer to the tables and figures in-line throughout the text13. 26 check resources on the Moodle course for exemplary lab reports 27 by each group; individual reports will not be accepted unless a prior exception has been granted 28 to: [email protected] | email subject line must include group # and day # 29 after lecture and/or during office hours (B257) 30 all references must be cited in the APA format 30 Lab report preparation (L2M) 6. Please interpret and describe your results in the discussion section. Avoid simple repetition of the results as described in the results section. In the discussion section, the results are expected to be compared with other literature results and corresponding deviations have to be critically discussed. The following information needs to be included in the lab reports: 1. Introduction (Day 1) Pipette calibration, mass of the 10 different water transfers including the calculation of the variance and standard deviation. Graphs containing the calibration curves for BSABradford as well as for the 4-nitrophenol. Best dilution of the enzyme for the CBH-I assay and calculated enzyme activities. Protein content of the chosen dilution of the CBH-I. 2. FPLC (Day 2) Graphs including all obtained FPLC-Chromatograms. Detailed purification table for CBH-I purification. 3. Polyacrylamide gel electrophoresis (Day 3) Figure containing the SDS-PAGE of the crude and purified CBH-I. Figure containing the Native-PAGE of the crude and purified CBH-I. Discuss the differences between the SDS- and Native-PAGE. Estimate the molecular mass obtained by SDS-PAGE. Conduct a literature search for reported CBH-I molecular masses and discuss them. 4. Enzyme kinetics (Day 4) Graphs including the linearization of the obtained enzyme activities according to the method of Lineweaver-Burk, Hanes-Wolf, Eadie-Hofstee and Michaelis-Menten. Graphs including the calculation of the Km and Vmax values deploying non-linear regression (without inhibitor) using Microsoft Excel. 31 Lab report preparation (L2M) Table containing the Km, Vmax, and Ki values calculated by the different linearization methods. 32 Appendix 1 (L2M) Protein quantification – Bradford – Instruction for use 33 Appendix 1 (L2M) 34 Appendix 2 (L2M) Z-correction factors for distilled water as a function of test temperature and air pressure31. How to use the z-correction factor table? Determine the correct Z-factor by finding the intersection between temperature and air pressure (assume air pressure to be 1 ATM/101.3 kPa). Round up the temperature value. For example, the Z-factor at 20.7 °C and 101.3 kPa is 1.0030. 31 Z-factors-calibration_2017_01_26.docx (Integra Biosciences) 35 Appendix 3 — ÄKTA™ Start (L2M) 36 Appendix 4 — ÄKTA™ Go (L2M) 37 Appendix 5 (L2M) Example calculation for PAGE sample preparation (hypothetical values) Volumetric capacity of 1 well32 is ~66 μL. Never ever fill up to full capacity. 1. Total volume of sample that you want to load in the well: 35 μL 2. Total protein (purified or crude sample) that you want to load in the well: 5 μg 3. Starting protein concentration of your sample: 0.2 μg*μL-1 4. Starting concentration of sample loading buffer (SLB): 6X 5. Required concentration of SLB: 1X 6. Volume of 6X SLB required to get 1X SLB: (35 μL) * (1X) ÷ 6X = ~5.83 μL 7. Volume remaining to add 5 μg of purified sample in: 35 μL - 5.83 μL = 29.17 μL 8. Required concentration of purified sample: 5 μg ÷ 29.17 μL = ~0.172 μg*μL-1 9. How to dilute the purified sample: C1 ∗ V1 = C2 ∗ V2 (0.2 μg*μL-1) * (V1) = (0.172 μg*μL-1) * (29.17 μL) V1 = ~25 μL in a microfuge tube, you need to mix 25 μL of your sample protein with 5.83 μL of SLB (6X) and 4.17 μL buffer to ensure that you load 5 μg protein in a well 10. Mix the above solution via vortex, centrifuge, and heat at 95°C for 5 min. Shortly cool on ice, mix, centrifuge, and then load into the well 32 one well in a 10-well comb (1.5 mm) 38  Appendix 6 (L2M) Protocol Bulletin 6210 Molecular Weight Estimation Run the standards and samples on an SDS-PAGE gel. 1 2 3 4 5 6 7 8 Top of resolving gel Process the gel with the desired stain and then destain to visualize the protein bands. Determine the Rf graphically or using Quantity One® analysis software (or equivalent). MW, kD Migration distance 250 of unknown band Using a ruler, measure the migration distance (45 mm) 1 150 from the top of the resolving gel to each standard band and to the dye front. 100 75 Migration distance For each band in the standards, calculate the of dye front 2 (67 mm) Rf value using the following equation: 50 Rf = migration distance of the protein/migration 37 distance of the dye front 25 Unknown band 20 Repeat this step for the unknown bands in 3 the samples. 15 10 Use a graphing program, plot the log (MW) 4 as a function of Rf. Fig. 1. Example showing MW determination of an unknown protein. Lane 1, 10 μl of Precision Plus Protein™ unstained standards; lanes 2–8, Generate the equation y = mx + b, and solve for y a dilution series of an E. coli lysate containing a hypothetical unknown protein 5 (GFP). Proteins were separated by SDS-PAGE in a Criterion™ 4–20% Tris-HCI to determine the MW of the unknown protein. gel and stained with Bio-Safe™ Coomassie stain. Gel shown is the actual size. 3.0 Standards Unknown 2.0 log MW 1.0 y = –1.9944x + 2.7824 r 2 = 0.997 0 0 0.2 0.4 0.6 0.8 1.0 Rf Fig. 2. Determining the MW of an unknown protein by SDS-PAGE. A standard curve of the log (MW) versus Rf was generated using the Precision Plus Protein standards from Figure 1. The strong linear relationship (r 2 > 0.99) between the proteins’ MW and migration distances demonstrates exceptional reliability in predicting MW. 39