Pipetting & Preparing Dilute Solutions PDF

Pipetting & Preparing Dilute Solutions https://www.youtube.com/watch?v=NgosWmRjjAo https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/Pipetting/ Q1: Convert 2 mg/mL to ug/uL. Show your solution. Q1: Convert 2 mg/mL to ug/uL. Show your solution. 2 mg mL x 1000 ug 1 mg x 1 mL = 2 ug 1000 uL uL Q2:...

Pipetting & Preparing Dilute Solutions https://www.youtube.com/watch?v=NgosWmRjjAo https://meschedl.github.io/MESPutnam_Open_Lab_Notebook/Pipetting/ Q1: Convert 2 mg/mL to ug/uL. Show your solution. Q1: Convert 2 mg/mL to ug/uL. Show your solution. 2 mg mL x 1000 ug 1 mg x 1 mL = 2 ug 1000 uL uL Q2: Convert 2 mg/mL to ug/mL. Show your solution. Q2: Convert 2 mg/mL to ug/mL. Show your solution. 2 mg mL x 1000 ug 1 mg x 1 mL 1 mL 2 mg mL x 1000 ug 1 mg = 2000 ug mL = 2000 ug mL C1V1=C2V2 → Use this formula to prepare a dilute solution if you have a target concentration and final volume for the new solution Q3: What volume of BSA stock solution do I need if I want to prepare 500 uL of 2 ug/uL BSA solution and my BSA stock solution has a concentration of 10 ug/uL? Desired BSA concentration: 2 ug/uL Desired final volume for new BSA concentration: 500 uL BSA stock solution concentration: 10 ug/uL Volume of BSA stock solution to be used: ? Q3: What volume of BSA stock solution do I need if I want to prepare 500 uL of 2 ug/uL BSA solution and my BSA stock solution has a concentration of 10 ug/uL? Desired BSA concentration: 2 ug/uL Desired final volume for new BSA concentration: 500 uL BSA stock solution concentration: 10 ug/uL Volume of BSA stock solution to be used: ? Formula derivation: V1 = C2V2 / C1 V1 = (2 ug/uL)(500 uL) / (10 ug/uL) = 1000 ug / 10 ug/uL V1 = 100 uL of BSA stock solution + 400 uL of buffer (e.g., PBS, grinding buffer, etc.) Dilution Factor → Refers to how many times less concentrated the dilute solution is compared to the original solution Using example in Q3: Dilute solution → 100 uL of BSA stock solution + 400 uL buffer (Final volume = 500 uL) Dilution factor = volume of solute + volume of diluent volume of solute Dilution factor = 100 uL solute + 400 uL buffer = 500 uL 100 uL solute 100 uL = 5, or 1:5 Dilution Factor → Refers to how many times less concentrated the dilute solution is compared to the original solution Q4: 50 uL of unknown protein sample was added to 550 uL of grinding buffer. What is the dilution factor? Dilution factor = volume of solute + volume of diluent volume of solute Dilution factor = 50 uL solute + 550 uL buffer = 600 uL 50 uL solute 50 uL = 12, or 1:12 → Dilute solutions can also be prepared using a desired DF (instead of a desired final concentration) and desired final volume Q5: Prepare a 750 uL solution of a 1:150 dilution Dilution factor = Final volume / Solute volume 150 = 750 uL / Solute volume Solute volume = 750 uL / 150 Solute volume = 5 uL of the stock solution + 745 uL of buffer or dH20 → Dilute solutions can also be prepared using a desired DF (instead of a desired final concentration) and desired final volume Q5: Prepare a 750 uL solution of a 1:150 dilution Solute volume = 5 uL of the stock solution + 745 uL of buffer or dH20 Note: If you know the concentration of the original solution, you can also get the concentration of the new dilute solution by dividing the initial concentration by the dilution factor (DF) Ex. If concentration of stock solution = 300 ug/uL Concentration of dilute solution = 300 ug/uL / 150 = 2 ug/uL → If you know the concentration of the original solution, you can also get the concentration of the new dilute solution by dividing the initial concentration by the dilution factor (DF) → The target final volume for the intermediate dilution (in this example, 20 uL) is arbitrary Step Dilutions Q6: Prepare a 400 uL solution of a 1:4000 dilution Needed volume is too small Traditional one-step preparation: 0.1 uL Dilution factor = Final volume / Solute volume 4000 = 400 uL / Solute volume Stock solution Solute volume = 400 uL / 4000 Solute volume = 0.1 uL of the stock solution + 399.90 uL of buffer or dH20 Desired solution → DF = 4000 → Final volume = 400 uL Step Dilutions Q6: Prepare a 400 uL solution of a 1:4000 dilution Using step dilution (Select 2 or more smaller dilution factors whose product is your target larger dilution factor): 4 uL 25 uL Stock solution Intermediate solution → DF = 40 → Final volume = 1000 uL Note: The target final volume of the intermediate solution (In this example, 1000 uL) is arbitrary Desired solution → DF = 100 (Overall DF with respect to stock solution is 4000) → Final volume = 400 uL Bradford assay Bradford assay ▪ Colorimetric protein quantitation assay ▪ Dye used is the Coomassie Brilliant Blue G-250 Source: https://130.15.90.125/BioLab/spectrophotometry/3_2P.html pH < 1: Dye has + charge (cation); red/brown color 1 < pH < 2: Dye has no net charge; green color pH > 2: Dye has – charge (anion); blue color → Binding of dye to protein stabilizes the anionic, blue form of the dye → Upon initial contact, CBB G-250 donates electrons to the protein, causing a change in the folding of the protein that exposes the protein’s hydrophobic regions o The change in protein conformation allows the dye to further bind to the protein through hydrophobic interactions and Van der Waals interactions o Also, there is electrostatic attraction between sulfonic groups of the dye (SO3-) and positive amine groups of amino acids Source: bio.libretexts.org Bradford assay ▪ Colorimetric protein quantitation assay ▪ Dye used is the Coomassie Brilliant Blue G-250 Wavelength of maximum absorption of the dye when it is not yet bound to protein → Bradford reagent is prepared with methanol (or ethanol) and glacial acetic acid Source: https://www.thermofisher.com/ph/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resource-library/pierceprotein-methods/chemistry-protein-assays.html Bradford assay ▪ Colorimetric protein quantitation assay ▪ Dye used is the Coomassie Brilliant Blue G-250 → Bradford reagent is prepared with methanol (or ethanol) and glacial acetic acid Source: https://www.thermofisher.com/ph/en/home/life-science/proteinbiology/protein-biology-learning-center/protein-biology-resource-library/pierceprotein-methods/chemistry-protein-assays.html Bradford assay ▪ Protein samples – Concentrations are unknown ▪ Protein standard (e.g. Bovine serum albumin (BSA)) – Multiple solutions with different known concentrations Protein Sample Protein Standard (i.e., BSA) Concentration (ug/uL) (x) Absorbance (y) Sample Absorbance (y) 0 0.0012 A 0.2334 0.25 0.0716 B 0.2137 0.5 0.1401 C 0.2786 0.75 0.2109 D 0.2772 1.0 0.2902 Bradford assay ▪ Protein samples – Concentrations are unknown ▪ Protein standard (e.g. Bovine serum albumin (BSA)) – Multiple solutions with different known concentrations Protein Standard (i.e., BSA) Concentration (ug/uL) (x) Absorbance (y) 0 0.0012 0.25 0.0716 0.5 0.1401 0.75 0.2109 1.0 0.2902 y = 0.2869x – 0.0007 x = (y + 0.0007) / 0.2869 For sample A: x = (0.2334 + 0.0007) / 0.2869 = 0.8160 ug/uL Bradford assay ▪ Protein samples – Concentrations are unknown ▪ Protein standard (e.g. Bovine serum albumin (BSA)) – Multiple solutions with different known concentrations Source: https://ecampusontario.pressbooks.pub/biochem2l06/chapter/3-1-laboverview-and-background-information/ UV-visible spectrophotometer → Measures absorbance or transmittance of light in the ultraviolet range (185–400 nm) and visible range (400-700 nm) of the electromagnetic radiation spectrum → Sample is placed into a cuvette, which is then placed inside the UV-vis spectrophotometer → Light of a specific wavelength passes through the sample at a specified path length (cuvette width), and the absorbance of the sample is measured UV-visible spectrophotometer → At 595 nm, the protein-bound dye absorbs orange light strongly, and the protein solution appears blue → The more dye molecules bound to proteins, the more orange light absorbed, and the more bluish the protein solution Source: https://www.pharmaceutical-int.com/article/bradford-proteinassay-evaluation-software.html Source: https://www.acs.org/content/acs/en/education/resources/highschool/chemmatters/pastissues/2015-2016/october-2015/food-colorings.html → The relationship between concentration and absorbance is given by the Beer-Lamber law Beer-Lambert law – States that absorbance is directly proportional to concentration, molar absorption coefficient and optical path length Transmittance Absorbance Incident light, or initial light intensity (I 0) Transmitted light, or final light intensity (I) → The relationship between concentration and absorbance is given by the Beer-Lamber law Beer-Lambert law – States that absorbance is directly proportional to concentration, molar absorption coefficient and optical path length Transmittance Absorbance Incident light, or initial light intensity (I 0) Transmitted light, or final light intensity (I) -To convert a given absorbance back to transmittance: 1 / Antilog [Absorbance] Beer-Lambert law – States that absorbance is directly proportional to concentration, molar absorption coefficient and optical path length y = mx + b Absorbance = (Molar absorptivity*optical path length)Concentration + b Points to remember → Proteins must be at least 3000 daltons/3 kilodaltons to be detected by the Bradford assay (1 dalton (Da) = 1 g/mol; 1 amino acid is around 110 Da) → Absorbance measured with Bradford assay is linear up to 2000 ug/mL Points to remember → If absorbance of sample is higher than the absorbance of the standard solution with the highest concentration, a) the sample should be diluted or b) another standard solution with a higher concentration should be prepared → The presence of surfactants/detergents (i.e., Sodium dodecyl sulfate (SDS)) in the protein solution causes precipitation of CBB G-250, interfering with dye-protein complex formation → Absorbance should be measured immediately (< than 1 hour) after mixing the dye with the protein sample because the dye-protein complex also tends to precipitate → New standard solutions for standard curve preparation should be prepared for new samples This sample is outside the range of the prepared standard solutions Points to remember → The sensitivity of CBB G-250 varies from protein to protein. Consequently, the standard curves made using the same concentrations of 2 different proteins will not be the same o 2 common standard proteins: bovine serum albumin (BSA) or bovine γ-globulin (BGG) Note: In any protein assay, the best protein to use as a standard is a purified preparation of the protein being assayed. In the absence of such an absolute reference protein, another protein must be selected as a relative standard. The best relative standard to use is the one that gives a color Source: https://assets.thermofisher.com/TFSyield similar to that of the protein being assayed. Assets/LSG/manuals/MAN0011181_Coomassie_Bradford_Protein_Asy_UG.pdf Points to remember → Store Bradford reagent away from light. Also, incubation of protein solutions after mixing with the Bradford reagent should be done away from light. → R or r (given by the lab’s spectrophotometer) – correlation coefficient: measures the strength and direction of the linear relationship between two variables, ranging from -1 to 1 R2 or r2 (given by MS Excel) – coefficient of determination: quantifies the proportion of the variance in the dependent variable (y) that is predicted or explained by the independent variable (x), ranging from 0 to 1 Other protein quantitation techniques Absorbance measurement at 280 nm Other protein quantitation techniques Colorimetric copper-based assays o Biuret Assay, Lowry Assay, & Bicinchoninic Acid (BCA) Assay Biuret Assay → Proteins with at least 3 amino acid residues form a complex with Cu2+ in an alkaline environment, consequently reducing Cu2+ to Cu1+ → The reaction produces a faint blue to purple color. The intensity of the color produced, measured at 540 nm, is directly proportional to protein concentration → Can detect higher protein concentrations than BCA Assay and Lowry Assay Other protein quantitation techniques Colorimetric copper-based assays BCA displaces proteins bound to Cu1+ BCA Assay → After the Biuret reaction, reduced copper (Cu1+ or cuprous ion) further reacts with bicinchoninic acid (BCA), resulting in an intense purple color measured at 562 nm (or any wavelength between 550 and 570 nm) → Can detect lower protein concentrations than the Biuret assay → Compatible with protein samples containing up to 5% surfactants/detergents Other protein quantitation techniques Colorimetric copper-based assays Biuret reaction Lowry Assay → After the Biuret reaction, a phosphomolybdic-phosphotungstic acid solution called Folin-phenol reagent is added and binds to the peptide-copper complex, producing an intense blue color, measured at 750 nm (or any wavelength between 650 nm and 750 nm) → Blue color is attributed to the heteropoly-molybdenum blue formed by the transfer of electrons from the peptide-copper complex to the phosphomolybdic-phosphotungstic acid complex → Can detect lower protein concentrations than the Biuret assay Homology Modeling of Protein Structure Homology modeling → Predicts the unknown three-dimensional structure of a protein by searching for another protein (Template) with known 3D structure whose sequence is highly similar to that of the unknown protein (Target or query) → Homologous or evolutionarily related proteins have similar sequences and similar 3D structure → Conservation of protein structure > conservation of amino acid sequence among homologues: even with only 30% sequence similarity, 2 proteins can still have similar 3D structure → Determining the structure of a protein is integral for understanding its function/s Homology modeling → Protein structure-function relationship is demonstrated in Sickle Cell Anemia Hemoglobin function is to bind oxygen. Mutant hemoglobin can still bind oxygen, but in low oxygen conditions, the mutant is prone to aggregation which causes deformation of red blood cells (where the hemoglobin protein is located) Levels of Protein Structure Levels of Protein Structure Amino acids Amino acids Tyr Primary structure → Peptide or Oligopeptide: Short chain of (2 to 20) amino acids → Polypeptide: Longer polymer with 50 amino acids or more, that can be in the folded state or not → Protein: Sometimes used interchangeably with polypeptide. Generally, however, this term refers to the folded, functional molecule composed of one polypeptide or multiple polypeptides Primary structure → Peptide bond is an amide bond formed between two amino acids through a dehydration synthesis reaction → N- to C-terminus synthesis: Proteins are always synthesized in a directional manner starting with the amine tail and ending with the carboxylic acid tail. New amino acids are always added onto the carboxylic acid tail, never onto the amine of the first amino acid in the chain. Primary structure → Peptide bond is an amide bond formed between two amino acids through a dehydration synthesis reaction → N- to C-terminus synthesis: Proteins are always synthesized in a directional manner starting with the amine tail and ending with the carboxylic acid tail. New amino acids are always added onto the carboxylic acid tail, never onto the amine of the first amino acid in the chain. Secondary structure → Local folded structures within a polypeptide formed by hydrogen bonding between atoms (Specifically, the C=0 group and the N-H group) of the peptide backbone → Side chains (R groups) are not involved in the formation of secondary structures Secondary structure R groups pointed away from the helix → Alpha-helix → Dotted lines show hydrogen bonds formed between C=O groups and N-H groups in the polypeptide backbone Secondary structure → Parallel and anti-parallel beta-pleated sheets Anti-parallel Parallel Secondary structure → Beta-pleated sheets Other less common secondary structures → Beta-turns, 310 helix, pi-helix → Beta-turn → Diameter: pi-helix > alpha-helix > 310 helix Tertiary structure → Overall 3D structure of the polypeptide → Tertiary structure is held together by different chemical interactions between 2 side chains or between a side chain and the peptide backbone atoms (with water molecules) (and negatively) Tertiary structure Tertiary structure Quaternary structure → The quaternary structure of a protein is formed by the assembly of multiple polypeptides, as in the case of antibody molecules (Shown below) o Homo-multimer/oligomer: a functional protein formed by 2 or more identical subunits or polypeptide chains o Hetero-multimer/oligomer: a functional protein formed by 2 or more different subunits or polypeptide chains → Hetero-multimeric protein Quaternary structure → The quaternary structure of a protein is formed by the assembly of multiple polypeptides o Homo-multimer/oligomer: a functional protein formed by 2 or more identical subunits or polypeptide chains o Hetero-multimer/oligomer: a functional protein formed by 2 or more different subunits or polypeptide chains → 4 different homo-multimeric proteins X-ray crystallography → Experimental technique considered as gold standard for determining 3D structure of proteins Protein crystal Ramachandran plot → Plot of the phi and psi angles of each amino acid of a protein o Phi angle (Φ): Angle between alpha-carbon and amino group o Psi angle (ψ): Angle between alpha-carbon and carboxyl group → Developed by Ramachandran, the plot shows the allowable phi and psi angles for amino acids so that the protein can form certain secondary structures PB Φ PB ψ Peptide bond (PB) Central or alpha carbon of this amino acid Ramachandran plot → Plot of the phi and psi angles of each amino acid of a protein o Phi angle (Φ): Angle between alpha-carbon and amino group o Psi angle (ψ): Angle between alpha-carbon and carboxyl group → Developed by Ramachandran, the plot shows the allowable phi and psi angles for amino acids so that the protein can form certain secondary structures → 1 dot = 1 amino acid in the chain Ramachandran plot → Plot of the phi and psi angles of each amino acid of a protein o Phi angle (Φ): Angle between alpha-carbon and amino group o Psi angle (ψ): Angle between alpha-carbon and carboxyl group → Developed by Ramachandran, the plot shows the allowable phi and psi angles for amino acids so that the protein can form certain secondary structures