BIOS 420L Cell Biology Concept Summary Exercise PDF

BIOS 420L Cell Biology Concept Summary Exercise 1-10 Proper Pipetting Techniques Pipetting is a crucial skill in laboratory work, especially in biochemistry and molecular biology, where precise and accurate measurement of liquids is often required. Proper pipetting techniques ensure reproducibilit...

BIOS 420L Cell Biology Concept Summary Exercise 1-10 Proper Pipetting Techniques Pipetting is a crucial skill in laboratory work, especially in biochemistry and molecular biology, where precise and accurate measurement of liquids is often required. Proper pipetting techniques ensure reproducibility and minimize errors in experiments. 1. Choosing the Right Pipette Pipette Selection: o Pipettes are available in different volume ranges, usually categorized as manual micropipettes, single-channel, and multi-channel pipettes. Choose the pipette that best matches the volume you intend to pipette. For example: ▪ P2, P10, P20 for volumes from 0.1 µL to 20 µL. ▪ P100, P200 for volumes from 20 µL to 200 µL. ▪ P1000 for volumes from 100 µL to 1000 µL (1 mL). Volume Range: Always use the pipette with a volume range that encompasses your desired volume. For example, use a P200 pipette for volumes ranging from 20 µL to 200 µL, but avoid using it for volumes close to its upper or lower limits to reduce error. 2. Setting the Correct Volume Adjusting the Volume: o Single-channel Pipettes: Turn the volume adjustment knob (typically located at the top or side of the pipette) to set the desired volume. The correct volume is typically displayed in a digital window, or in the case of mechanical pipettes, by aligning the marking on the barrel. o Multi-channel Pipettes: The volume is adjusted in the same way, but ensure all channels are set to the same volume. Correct Settings: Double-check the set volume by aligning the pipette with the appropriate scale markings to ensure accuracy. 3. Proper Pipetting Technique Using the Pipette Tip: o Attach a Clean Tip: Always use a new, sterile pipette tip for each sample to avoid contamination. o Securely Fit the Tip: The pipette tip should be securely attached to the pipette. An improper seal can cause inaccurate volume delivery. Pipetting Procedure: 1. Press the Plunger: Press the pipette plunger to the first stop, which is the point of resistance. This creates a vacuum that will draw the liquid into the pipette tip. 1 BIOS 420L Cell Biology Concept Summary Exercise 1-10 2. Immerse the Tip in the Liquid: Place the tip in the liquid just below the surface to avoid air bubbles, but not too deep to avoid contamination from the outer walls. 3. Draw the Liquid: Slowly release the plunger to aspirate the liquid. Avoid letting go of the plunger too quickly, as this can create bubbles and inaccurate measurements. 4. Dispensing the Liquid: ▪ Hold the pipette vertically and gently press the plunger to the first stop to expel the liquid. ▪ Press past the first stop to the second stop to ensure that all the liquid is expelled from the tip. ▪ Always dispense the liquid slowly to avoid splashing. Ejecting the Tip: Once dispensing is complete, press the ejector button to discard the tip into a waste container. 4. Tips for Accuracy and Precision Consistent Speed: Pipetting too quickly or too slowly can cause variation in the amount of liquid dispensed. Maintain a consistent and steady pace. Correct Angle: Pipette tips should be held vertically during both aspiration and dispensing. Holding them at an angle can cause errors due to liquid retention. Pre-wetting the Tip: To improve accuracy, aspirate and dispense the liquid back into the original container one or two times before pipetting the sample. This helps to condition the tip and reduce error from surface tension. 5. Maintenance and Calibration Regular Calibration: Regularly calibrate your pipettes using a balance to ensure accuracy. Poorly calibrated pipettes can lead to inconsistent results. Routine Maintenance: Clean and maintain your pipettes regularly to ensure smooth operation. Check for any signs of damage or wear (e.g., cracked tips or malfunctioning plunger). Common Mistakes in Using Pipettes 1. Incorrect Volume Setting: Setting the wrong volume, especially on adjustable pipettes, is a common mistake. Always double-check the volume before use. 2. Using the Wrong Pipette for the Volume: Using a pipette that is not suited for the volume you are working with can introduce error. Avoid using a P1000 for small volumes like 10 µL, as this reduces precision. 3. Not Pre-wetting the Tip: Failing to pre-wet the pipette tip can lead to inaccurate volume measurements, as the surface tension of the liquid can cause loss during aspiration and dispensing. 2 BIOS 420L Cell Biology Concept Summary Exercise 1-10 4. Plunger Release Too Quickly: Releasing the plunger too quickly can create air bubbles in the tip, leading to inaccurate measurements. 5. Not Changing Tips Between Samples: Reusing tips without proper cleaning can cause contamination between samples. Always change tips between different liquids, samples, or reagents. 6. Incorrect Angle of Pipetting: Holding the pipette at an angle or dipping the tip too deep can result in inaccurate measurements or contamination. 7. Pipetting Too Quickly or Too Slowly: Rapid pipetting can cause liquid splashing or inaccurate measurements, while very slow pipetting can lead to inconsistent results. Aim for a controlled, steady pace. 8. Touching the Tip: Touching the tip of the pipette to the side of a container can result in liquid clinging to the wall, leading to inaccuracies in volume. 9. Incorrect Dispensing: Not dispensing the full volume or pressing the plunger too forcefully can result in incomplete liquid transfer. Always ensure the plunger is pressed to the second stop for total volume transfer. Exercise 1: Protein Assay and Standard Curve Key Components and Their Roles 1. Bovine Serum Albumin (BSA) Role: BSA serves as a standard protein to create a calibration curve. The protein concentration range (0.2 mg/ml to 1.8 mg/ml) provides a series of known values that allow for the estimation of protein concentration in unknown samples. Significance: BSA is commonly used as a standard because it is abundant, relatively inexpensive, and behaves similarly to other proteins in colorimetric assays. It ensures consistent and reliable protein measurements across experiments. 2. Pierce 660 nm Protein Assay Reagent Role: The 660 nm reagent contains a dye-metal complex (typically a polyhydroxybenzenesulfonephthalein-type dye combined with a transition metal ion, like molybdenum or vanadium) that binds to proteins in solution. The dye changes color when it interacts with proteins, producing a green color shift, which can be quantified by measuring absorbance at 660 nm. Significance: This reagent allows for a rapid, sensitive, and linear measurement of protein concentration in a wide range (25 µg/ml to 2000 µg/ml). The colorimetric change is proportional to the amount of protein present, making it easy to quantify unknown samples by comparison to a standard curve. 3. Microplate and Microplate Reader 3 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Role: The microplate (typically 96 wells) is used to hold small volumes of protein standards, unknown samples, and reagents. A microplate reader measures the absorbance of each well at 660 nm, providing the data necessary to calculate protein concentrations. Significance: The microplate format allows for high-throughput analysis of multiple samples simultaneously, making it ideal for experiments where many protein samples need to be tested. The reader's precise absorbance measurement at 660 nm enables accurate detection of the color change produced by the protein assay. Chemical Reactions and Mechanism The Pierce 660 nm protein assay reagent forms a dye-metal-protein complex in acidic solution, where the dye binds to protein molecules through electrostatic interactions and hydrophobic interactions. Upon binding, the dye changes from its initial reddish hue (λmax = 450 nm) to a green color (λmax = 660 nm), indicating the presence of protein. Absorbance at 660 nm is proportional to the protein concentration. This colorimetric change is directly measurable using the microplate reader, which detects the light absorption and converts it into a digital reading that can be used to calculate protein concentration based on the standard curve. Standard Curve Construction Standard Curve Role: The standard curve is crucial for translating absorbance readings into protein concentrations. By plotting the absorbance (A660) of known concentrations of BSA (standards) against their concentrations (mg/ml), a linear relationship is established. This curve is used to determine the protein concentration of unknown samples by comparing their absorbance values to the curve. Significance: The standard curve helps compensate for any variabilities in assay conditions (e.g., slight changes in reagent quality or environmental factors), ensuring that protein concentrations can be accurately determined across a wide range of samples. Significance of Assay Reagents and Wavelength 660 nm Absorbance: The choice of 660 nm for measurement is significant because it provides optimal sensitivity and specificity for detecting the green color shift in the dye- metal-protein complex. This wavelength avoids interference from other substances that may be present in biological samples. 4 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Color Shift: The shift from reddish (450 nm) to green (660 nm) upon protein binding ensures that the assay can be sensitive to low protein concentrations, making it suitable for a wide variety of applications where protein levels need to be measured with precision. Experimental Significance in Quantification Accurate Measurement: The ability to create a standard curve and compare absorbance values from unknowns ensures that protein quantification is both accurate and reproducible. This allows researchers to know exactly how much protein is in a given sample, which is essential for maintaining experimental consistency. Rapid and Efficient: The 660 nm assay is advantageous for its speed and ease of use compared to other protein assays (like Bradford or Lowry), making it particularly useful in high-throughput settings where time and efficiency are crucial. Quality Control Blank Control: Water (blank) is used to correct for any baseline absorbance in the assay, ensuring that only the absorbance due to the protein-dye interaction is measured. The blank accounts for the reagent’s inherent absorbance and any container or plate material effects. Replicates: Using replicate measurements for both standards and unknowns ensures precision and helps detect errors in pipetting or reagent preparation. This increases the reliability of the results. Exercise 2: Tissue Printing 1. Purpose and Significance of the Experiment Purpose: Tissue printing is used to localize specific proteins and enzymes (such as peroxidase and polyphenol oxidase) in plant and fungal tissues. By transferring proteins from tissue sections onto PVDF (Polyvinylidene Fluoride) membranes and applying colorimetric detection methods, researchers can visualize the distribution of enzymes and total proteins within plant tissues. Significance: Understanding where enzymes like peroxidase and polyphenol oxidase are located in tissues can provide insights into metabolic pathways, defense mechanisms, and growth processes in plants. The experiment also demonstrates the use of protein blotting techniques, which are fundamental tools in molecular biology and biochemistry (e.g., Western blotting). 2. Key Chemicals and Their Roles Hydrogen Peroxide (H₂O₂): 5 BIOS 420L Cell Biology Concept Summary Exercise 1-10 o Role: Acts as a substrate for peroxidase. The enzyme catalyzes the breakdown of H₂O₂, producing a color change when reacted with 4-chloro-1-naphthol. This reaction allows for the localization of peroxidase in the tissue. o Significance: Hydrogen peroxide is essential for the activity of peroxidase, an enzyme that is involved in plant defense and lignin biosynthesis. 4-Chloro-1-naphthol Solution: o Role: This chemical undergoes a color change when oxidized by peroxidase in the presence of hydrogen peroxide. The reaction produces a blue-colored product that can be easily detected on the tissue blot. o Significance: The colorimetric change allows for the visualization and localization of peroxidase activity in plant tissues, which is often found in the epidermis and vascular tissues, particularly in relation to lignification. Catechol and MBTH (3-Methyl-2-benzo-thiazolinone hydrazine): o Role: These chemicals are used for detecting polyphenol oxidase (PPO) activity. Polyphenol oxidase catalyzes the oxidation of catechol, producing an intermediate that reacts with MBTH to form a colored product. o Significance: This reaction allows for the localization of polyphenol oxidase, which is involved in polyphenol metabolism and defense responses in plants, contributing to browning in fruits and vegetables. Ponceau S: o Role: Ponceau S is used to stain total proteins on the PVDF membrane, providing a general overview of protein distribution across the tissue sections. o Significance: This stain is important for visualizing the overall protein content and comparing it to the localized enzyme activities (peroxidase and polyphenol oxidase), which can help in understanding the relationship between enzyme distribution and protein expression in the tissues. PVDF Membranes: o Role: PVDF (Polyvinylidene Fluoride) membranes are used as the medium for protein transfer during tissue printing. They have a high affinity for proteins and are often used in various blotting techniques (e.g., Western blotting) for detecting and localizing proteins. o Significance: PVDF membranes are ideal for transferring proteins from tissue sections because they bind proteins efficiently, allowing them to be detected with various staining methods. They also provide high-quality results for visualizing proteins in biological samples. 6 BIOS 420L Cell Biology Concept Summary Exercise 1-10 3. Principle of Tissue Printing Tissue Printing: This technique involves pressing thin sections of plant or fungal tissues onto PVDF membranes, which transfer the proteins and enzymes present in the tissue onto the membrane. The proteins are then localized using specific colorimetric assays for enzymes like peroxidase and polyphenol oxidase or total protein staining. Significance: Tissue printing allows for the visualization of protein localization without the need for complex immunohistochemistry techniques. This is a simpler, cost-effective method to explore spatial distribution of enzymes and proteins within tissues, aiding in research on metabolic processes, defense mechanisms, and developmental biology. 4. Comparison of Peroxidase, Polyphenol Oxidase, and Total Protein Distribution Peroxidase Localization: o Location: In plants, peroxidases are typically found in the epidermis and some vascular elements, playing a key role in oxidative stress responses and lignin biosynthesis. o Color Reaction: The blue color produced by the reaction between peroxidase, hydrogen peroxide, and 4-chloro-1-naphthol marks the areas where peroxidase is active in the tissue. Polyphenol Oxidase (PPO) Localization: o Location: Polyphenol oxidase is involved in polyphenol metabolism, and its distribution can be observed in areas involved in defense responses (e.g., in response to wounding or stress). o Color Reaction: The reaction between catechol and MBTH produces a color change that highlights PPO activity, typically in areas where polyphenols are synthesized or accumulated. Total Protein Localization: o Ponceau S Staining: This method stains all proteins present in the tissue sections, giving a general overview of protein distribution. It allows for the comparison of enzyme localization (peroxidase and PPO) with total protein content. o Significance: The Ponceau S stain serves as a control for total protein content, enabling comparison with the localized enzyme activities. This can help identify whether specific enzymes are concentrated in particular regions of the tissue. 5. Concept of Protein Localization in Plant Tissues 7 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Peroxidase: Peroxidases are primarily involved in breaking down hydrogen peroxide, a byproduct of cellular metabolism, thus protecting plant cells from oxidative damage. They are often found in cell wall structures and vascular tissues involved in lignin biosynthesis. Polyphenol Oxidase (PPO): Polyphenol oxidase plays a critical role in the oxidation of phenolic compounds and is involved in plant defense mechanisms, particularly in response to wounding or pathogen attack. The localization of PPO can reveal areas of high polyphenol activity in the plant, often in the epidermis or vascular tissues. Total Protein: The total protein stain (Ponceau S) helps in visualizing the overall protein content in the tissues. By comparing the distribution of total proteins to specific enzymes (peroxidase, PPO), we can infer the role of different proteins in the plant’s metabolic processes. 6. Practical Considerations and Key Observations Membrane Handling: PVDF membranes must be handled with care because they are hydrophobic and require activation by methanol before use. This ensures that proteins will bind to the membrane. Blotting Procedure: It's crucial to ensure that the tissue sections are pressed evenly onto the PVDF membrane for a uniform transfer of proteins. Any variation in pressure or contact time may lead to inconsistent protein transfer. Staining and Detection: Different chemicals used for enzyme staining (e.g., 4-chloro-1- naphthol for peroxidase and MBTH for PPO) produce color changes that can be visually compared, allowing researchers to identify the localization of enzymatic activity within the tissue sections. 7. Conclusion and Data Interpretation Comparing Enzyme Distributions: The distribution of peroxidase and polyphenol oxidase can reveal differences in enzyme localization across different plant species and tissues. For instance, peroxidase might be more concentrated in vascular tissues involved in lignin formation, while polyphenol oxidase might be found in the epidermis or other regions where polyphenols are synthesized. Use of Total Protein Staining: By comparing enzyme activity with total protein distribution, researchers can understand how specific enzymes contribute to the overall protein profile of the plant or fungal tissues. Exercise 3: Cytosolic Lactate Dehydrogenase (LDH) 1. Introduction: Lactate Dehydrogenase (LDH) 8 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Significance: LDH is a key enzyme in cellular metabolism, catalyzing the reversible conversion of pyruvate to lactate with the reduction of NADH to NAD+. It is important for the regeneration of NAD+ in anaerobic conditions. Isozymes: LDH exists as different isozymes (e.g., H4, M4, H3M, H2M2), which are tissue- specific. For example, H4 is predominant in heart tissue, while M4 is found in muscle and liver. Subunit Structure: LDH is a tetramer composed of two types of subunits, H and M, each with a molecular weight of 35,000 daltons. The isozyme composition reflects the enzyme's functional specialization in different tissues. 2. Enzyme Assay Method Significance: The spectrophotometric assay measures the activity of LDH by tracking the decrease in NADH absorbance at 340 nm, which correlates with the enzyme’s catalytic activity. Reaction: LDH catalyzes the conversion of pyruvate to lactate, using NADH as a coenzyme: Pyruvate + NADH ↔ Lactate + NAD+. Key Role of Chemicals: o NADH (6.6 mM): Serves as the electron donor, being oxidized to NAD+ during the reaction. o Sodium Pyruvate (30 mM): Acts as the substrate for the LDH-catalyzed reaction. o Tris-HCl Buffer (0.2 M, pH 7.3): Maintains the pH of the reaction for optimal enzyme activity. 3. Homogenization and Enzyme Extraction Purpose: To extract LDH from liver cells for assay. This involves breaking open the cells and separating the soluble cytosolic fraction from cellular debris. Homogenization Buffer (0.15 M NaCl): Used to wash the liver tissue and provide an isotonic environment, preventing cell rupture and loss of enzyme activity. Centrifugation (20,000g): The homogenate is centrifuged to remove cell organelles and debris, with the supernatant containing the soluble cytosolic LDH. Significance of Differential Centrifugation: Helps separate cellular components by size and density. The high-speed centrifugation ensures that only soluble proteins (like LDH) remain in the supernatant. 4. Protein Assay (Pierce 660 nm Protein Assay) Significance: Quantifies the protein concentration in the enzyme extract, which is essential for determining the specific activity of LDH. 9 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Role of Reagents: The 660 nm reagent binds to proteins, forming a complex that shifts absorbance to 660 nm. The absorbance correlates with protein concentration. Bovine Serum Albumin (BSA) Standard: Used to create a standard curve, allowing the quantification of protein concentration in unknown samples. 5. Spectrophotometric Assay of LDH Activity Significance: The rate of NADH oxidation is measured spectrophotometrically to determine LDH activity. Calculation of Enzyme Activity: o Absorbance (A340): The decrease in absorbance at 340 nm is used to determine the enzyme activity. o Specific Activity (μmoles/min/mg protein): The enzyme activity is normalized to the protein content, which allows for comparison across different samples. Molar Extinction Coefficient of NADH: A known constant (6,220 M^-1cm^-1) is used to convert changes in absorbance (A340) into enzyme activity in micromoles per minute. 6. Calculation of Specific Activity of LDH Significance: The specific activity reflects the enzyme's catalytic efficiency and is a measure of the enzyme purity or concentration relative to the total protein in the extract. Exercise 4: ELISA Introduction: Antibodies, Antigens, and ELISA Antibodies: Proteins produced by the immune system that bind specifically to antigens (foreign molecules). Antigens: Foreign molecules, such as viruses or bacteria, that stimulate an immune response. ELISA (Enzyme-Linked Immunosorbent Assay): A diagnostic test that detects the presence of antigens or antibodies using enzyme-linked antibodies. ELISA Process Overview 1. Antigen Coating o Antigen (IgY) is coated on microplate wells. o Significance: Anchors the antigen to the surface for antibody interaction. Blocking with Tween 20 prevents non-specific binding. 2. Primary Antibody Binding 10 BIOS 420L Cell Biology Concept Summary Exercise 1-10 o Primary antibody (rabbit anti-chicken IgY) binds to the antigen. o Significance: Ensures antigen-specific detection. The primary antibody is the first level of target recognition. 3. Secondary Antibody Binding o Secondary antibody (HRP-conjugated) binds to the primary antibody. o Significance: Amplifies the signal by linking the primary antibody to HRP, which catalyzes the color change. 4. Chromogenic Reaction o TMB substrate is added, which reacts with HRP to produce a blue color. o Significance: Color change indicates the presence of antigen. The intensity of color correlates with antigen concentration. 5. Washing Steps o Wash the wells to remove unbound reagents. o Significance: Ensures specificity by removing non-specific binding and reducing background noise. Data Analysis and Interpretation Controls: o Positive Controls: Known antigen-containing samples (should turn blue). o Negative Controls: No antigen, should remain colorless. o Blank Wells: Contain PBS only, used to account for background signal. Results Interpretation: o Positive Result: Wells that turn blue indicate the presence of the antigen. o Negative Result: Wells that remain colorless indicate no antigen. o Quantification: Absorbance at 450 nm correlates with the antigen concentration (higher absorbance = higher antigen levels). Exercise 5: Differential Permeability of Plasma Membrane Introduction: Plasma Membrane and Diffusion Erythrocytes (red blood cells) serve as model cells to study plasma membrane properties. 11 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Hemolysis (cell rupture) occurs when molecules diffuse across the plasma membrane, causing the cell to swell and eventually burst. Hemolysis Endpoint: Observed when the blood solution transitions from opaque to transparent. Parameters affecting permeability: 1. Molecular Weight: Larger molecules may have slower diffusion rates. 2. Lipid Solubility: Lipid-soluble molecules more easily cross the lipid bilayer. 3. Number of Polar Groups: Molecules with more polar groups may have different permeabilities due to interactions with the membrane. The Fluid Mosaic Model of the membrane (Singer and Nicolson, 1972) explains the dynamic nature of cell membranes with lipid bilayers interspersed with proteins, influencing selective permeability. Experiment: Effects of Molecular Weight, Lipid Solubility, and Polar Groups on Permeability 1. Effect of Molecular Weight on Permeability Objective: Investigate the relationship between molecular weight and the time it takes for hemolysis to occur. Hypothesis: Larger molecules (higher molecular weight) are expected to have slower diffusion rates across the membrane, leading to longer hemolysis times. 2. Effect of Lipid Solubility on Permeability Objective: Examine how lipid solubility (partition coefficient) affects permeability. Hypothesis: As lipid solubility (partition coefficient) increases, molecules are expected to diffuse more rapidly through the membrane, decreasing hemolysis time. 3. Effect of Number of Polar Groups on Permeability Objective: Investigate how the number of polar (hydroxyl) groups influences permeability. Hypothesis: Molecules with more hydroxyl groups (e.g., glycerol) are expected to have slower diffusion rates due to increased interaction with the aqueous environment, leading to slower hemolysis. Table 1: Molecular Weights Reagent Molecular Weight Urea 60.1 n-Propyl Alcohol 60.1 12 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Reagent Molecular Weight Ethylene Glycol 62.1 Glycerol 92.1 Glucose 180.0 Table 2: Partition Coefficient and Carbon Length Reagent Partition Coefficient # Carbons Methyl Alcohol 0.14 1 Ethyl Alcohol 0.26 2 n-Propyl Alcohol 1.90 3 Table 3: Number of Hydroxyl Groups Reagent # Hydroxyl Groups n-Propyl Alcohol 1 Ethylene Glycol 2 Glycerol 3 Conclusion Molecular Weight: Larger molecules have longer hemolysis times due to slower diffusion across the membrane. Lipid Solubility: Molecules with higher lipid solubility (higher partition coefficient) diffuse faster, leading to quicker hemolysis. Polar Groups: The presence of multiple hydroxyl groups reduces membrane permeability by increasing interaction with the aqueous environment, resulting in slower hemolysis. Exercise 6: Isolation of Plasma Membrane from Erythrocytes Introduction: Plasma Membrane and Membrane Proteins The plasma membrane from erythrocytes (red blood cells) was one of the first to be isolated and studied in detail (Steck, 1974). This is facilitated by the lack of internal membranes or organelles in mature erythrocytes. 13 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Isolation of Plasma Membranes: The erythrocyte plasma membrane can be isolated by lysing the red blood cells in hypotonic buffer followed by centrifugation, which results in the membrane pelleting out. The proteins in the plasma membrane are mostly hydrophobic, and these can be analyzed using SDS polyacrylamide gel electrophoresis (SDS-PAGE). This method separates proteins based on their molecular weight. Procedure Overview 1. Isolation of Plasma Membrane Centrifuge whole blood at high speed to pellet the cells. Wash the cells once with isotonic phosphate buffer and then resuspend them in hypotonic phosphate buffer to lyse the cells. Centrifuge the lysed cells at higher speed to pellet the plasma membrane. Resuspend the plasma membrane in hypotonic buffer, wash once, and then suspend in distilled water for analysis. 2. Observation of Isolated Membranes Prepare wet mounts of the isolated plasma membranes and observe them under a microscope to distinguish between intact erythrocytes and isolated membranes. 3. Analysis of Membrane Proteins by SDS-PAGE Dissolve a small amount of the isolated plasma membrane in 2X SDS sample buffer. Load the sample into SDS polyacrylamide gel and run electrophoresis. After electrophoresis, stain the gel with Coomassie Blue R-250 to visualize the proteins. Plot the mobility of standard molecular weight markers versus their molecular weight to determine the approximate molecular weight of the proteins in the plasma membrane. Key Concepts Isolation of Plasma Membranes Hypotonic Lysis: Red blood cells (RBCs) are lysed using a hypotonic buffer to break open the cells, releasing the plasma membrane. The centrifugation steps help separate the membrane from other cellular components. Analysis by SDS-PAGE SDS-PAGE is a powerful technique used to separate proteins by their molecular weight. 14 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Sodium dodecyl sulfate (SDS) binds to proteins, imparting a uniform negative charge, allowing the proteins to be separated solely based on size when an electric field is applied. After electrophoresis, the gel is stained with Coomassie Blue, which binds to proteins, making them visible. Graphing and Molecular Weight Determination Semi-log plot: A semi-logarithmic graph is used to correlate the mobility of the molecular weight markers (standard proteins) with their molecular weights. By comparing the migration distance of the unknown proteins to the standard curve, the molecular weight of plasma membrane proteins can be estimated. Data and Analysis Electrophoresis Results: After electrophoresis, you will obtain a gel showing bands corresponding to different membrane proteins. These bands can be used to estimate the molecular weight of the proteins based on the standard curve. Graph: A semi-logarithmic graph should be constructed with the molecular weight (log scale) on the y-axis and mobility (linear scale) on the x-axis to analyze the results. Exercise 7: Western Blot Key Concepts and Summary Western Blotting is a powerful technique used to identify specific proteins in a sample by using antibody-antigen interactions. It involves several key steps: protein separation (via SDS-PAGE), protein transfer to a membrane (PVDF), blocking to prevent non-specific binding, incubation with a primary antibody, and detection using a label (such as DyLight488). SDS-PAGE separates proteins based on size, ensuring that when transferred to the membrane, proteins are spatially resolved and can be probed individually with antibodies. The PVDF membrane is used for its high binding capacity and mechanical strength, which makes it ideal for handling multiple wash and detection steps. The blocking step ensures that antibodies bind only to the target protein, improving the specificity of the assay and reducing background noise. The primary antibody specifically binds to the target protein, and in this case, the DyLight488 fluorescent label allows for direct visualization without the need for a secondary antibody. Purpose of Western Blot: Western blot is used to detect specific proteins based on their size and their ability to bind to a 15 BIOS 420L Cell Biology Concept Summary Exercise 1-10 particular antibody. In this case, the objective is to identify whether unknown recombinant protein samples contain a His-tag. Reason for SDS-PAGE prior to transfer: SDS-PAGE separates proteins based on size (molecular weight). The SDS detergent denatures proteins and imparts a negative charge, allowing proteins to migrate through the gel. This step ensures that proteins are distributed according to size before being transferred to the membrane. PVDF Membrane and Its Role in Western Blotting: PVDF (polyvinylidene fluoride) membrane is used for transferring proteins from the gel due to its high protein-binding capacity. It is hydrophobic and resistant to multiple washes, which ensures that proteins bind securely and are not lost during the process. Importance of Blocking Step: Blocking prevents non-specific binding of antibodies to the membrane. By blocking unoccupied sites, it ensures that the antibody specifically binds only to the protein of interest, reducing background noise and improving the clarity of the signal. Consequences of Skipping the Blocking Step: Skipping the blocking step results in non-specific binding, which leads to high background signals. This makes it difficult to clearly identify the protein of interest, as false positives may occur, compromising the accuracy of the results. Role of Primary Antibody in Western Blotting: The primary antibody binds specifically to the protein of interest. In this exercise, the anti-His antibody is used to bind to proteins containing a His-tag, enabling their identification. Label Attached to the Antibody: The primary antibody in this exercise is conjugated to a DyLight488 fluorescent label. This fluorescent tag eliminates the need for a secondary antibody, simplifying the detection process and saving time. Exercise 8: Lysosomal Acid Phosphatase Assay Key Components and Their Roles Liver Tissue (Chicken Liver): Source of lysosomes, which are intracellular organelles responsible for digestion and waste processing. Bio-homogenizer: Used for breaking down liver tissue to release cellular contents, including lysosomes. Isolation Buffer (0.25 M sucrose, 0.01 M Tris, pH 7): Maintains the osmotic balance and pH during the extraction process to prevent damage to organelles. Acid Phosphatase Assay Buffer (0.15 M sodium acetate, pH 5): Provides the optimal pH environment for acid phosphatase activity, which is essential for the assay. 16 BIOS 420L Cell Biology Concept Summary Exercise 1-10 o-Carboxyphenylphosphate (CPP): Substrate used in the acid phosphatase assay. It is hydrolyzed by acid phosphatase to produce salicylic acid, which can be measured spectrophotometrically. Spectrophotometer: Measures the absorbance of salicylic acid at 300 nm to quantify enzyme activity. Protein Assay Reagents: Used to determine the protein concentration of the lysosomal preparation, which is necessary for calculating specific enzyme activity. Importance of Differential Centrifugation Principle of Differential Centrifugation: This technique separates cellular components based on their size, density, and shape by applying increasing centrifugal forces. o At low speeds (1,700 g), large cellular debris (e.g., unbroken cells, nuclei) is removed, leaving a supernatant containing smaller organelles. o At higher speeds (20,000 g), the denser lysosomes are pelleted, while other smaller organelles (e.g., ER fragments) remain in the supernatant. Significance: Differential centrifugation is crucial for isolating pure lysosomal preparations. It allows for the separation of lysosomes from other cellular organelles, which could contaminate the sample and interfere with subsequent analyses. Acid Phosphatase Assay Enzyme Target: Acid phosphatase is a key lysosomal enzyme involved in hydrolyzing phosphate monoesters at an acidic pH. Role of CPP: The substrate CPP is used in the assay because it is specifically hydrolyzed by acid phosphatase to release salicylic acid. The amount of salicylic acid produced correlates with the enzyme activity. Spectrophotometric Measurement: Salicylic acid absorbs light at 300 nm, making it easy to quantify the hydrolysis reaction using a spectrophotometer. This allows for accurate measurement of the enzyme's activity. Key Points to Remember Optimal pH for Acid Phosphatase: Acid phosphatase has an optimal activity at acidic pH (around pH 5), which is why the assay buffer is set to pH 5. Testing the enzyme activity at different pH values (e.g., pH 7) helps determine how pH affects enzyme function and provides insights into its physiological role. STUDY THE CALCULATION SHEET FOR FINAL EXAM 17 BIOS 420L Cell Biology Concept Summary Exercise 1-10 EXERCISE 9: Mitochondrial Fumarase Assay Purpose: The exercise focuses on isolating mitochondria from chicken liver using differential centrifugation, followed by an assay of fumarase activity, a mitochondrial enzyme involved in the citric acid cycle. The assay is used to confirm the presence and functionality of mitochondria in the isolated sample. Differential Centrifugation: This technique is central to isolating mitochondria from other cellular components. It involves sequential centrifugation at increasing speeds to separate organelles based on their size and density: Low-speed centrifugation (1,700 g): This step removes larger cellular debris such as unbroken cells and nuclei. High-speed centrifugation (20,000 g): The mitochondrial pellet is obtained at this stage, with mitochondria being pelleted (contaminated with lysosomes and peroxisomes) while other smaller organelles and cytoplasmic components remain in the supernatant. The purity of the mitochondrial fraction is critical for reliable enzyme assays, and differential centrifugation helps isolate mitochondria while minimizing contamination from other cell structures. Fumarase Assay: The isolated mitochondria are assayed for fumarase activity, an enzyme critical to the citric acid cycle. The activity is measured spectrophotometrically by detecting the production of fumarate from L-malate, with a measurable increase in absorbance at 250 nm. The rate of change in absorbance corresponds to the amount of fumarate produced, which is used to calculate enzyme activity. Protein Quantification: Protein concentration in the mitochondrial sample is determined using the Pierce 660 protein assay, which is essential for normalizing enzyme activity results and calculating the specific activity of fumarase. Key Steps and Purpose: Homogenization and Centrifugation: These steps are designed to break up liver tissue and separate mitochondria from other cellular components through size and density-based centrifugation. Resuspension and Protein Measurement: Ensuring the mitochondria are resuspended properly and protein content is quantified is important for assessing mitochondrial purity and normalizing assay data. Fumarase Assay: The assay measures the enzyme's activity as an indicator of mitochondrial function, with the spectrophotometric detection of fumarate production serving as the key method for activity measurement. Outcome: The key outcome of this exercise is the quantification of fumarase activity in the mitochondrial sample, which serves as evidence of successful mitochondrial isolation and 18 BIOS 420L Cell Biology Concept Summary Exercise 1-10 functional integrity of the isolated mitochondria. The specific activity of fumarase is used to assess both the presence and enzymatic activity of mitochondria in the sample. STUDY THE CALCULATION SHEET FOR FINAL EXAM EXERCISE 10: Peroxisomal Catalase Assay Purpose: The exercise involves isolating peroxisomes from turnip tissue and assaying their catalase activity. Catalase, an enzyme found in peroxisomes, breaks down hydrogen peroxide (H₂O₂), a toxic by-product of cellular metabolism. The goal is to measure the rate of hydrogen peroxide degradation to assess peroxisomal function. Differential Centrifugation for Peroxisomal Isolation: Homogenization: The turnip tissue is homogenized in a cold isolation buffer to break down the cell wall and release cellular components. Centrifugation: The crude peroxisomal pellet is obtained through sequential centrifugation at different speeds: o Low-speed centrifugation (1,700 g): Removes larger cell debris such as nuclei. o High-speed centrifugation (20,000 g): Pellets peroxisomes (contaminated with mitochondria) while other smaller organelles in the supernatant. Resuspension: The crude peroxisomal pellet is resuspended in assay buffer for enzyme activity measurement. This step ensures that the peroxisomes are available for the subsequent assay. Catalase Assay: The presence and activity of catalase in the isolated peroxisomes are measured by monitoring the disappearance of hydrogen peroxide (H₂O₂) at 240 nm. This decrease in absorbance indicates the breakdown of H₂O₂, which is the result of catalase activity. The molar extinction coefficient of H₂O₂ is used to calculate the rate of H₂O₂ degradation. The rate of catalase activity is then used to determine the total amount of hydrogen peroxide degraded per unit of tissue weight, providing insight into the functional capacity of the peroxisomal preparation. Key Steps and Purpose: Homogenization and Centrifugation: These initial steps are crucial for breaking open the cells and isolating peroxisomes from other organelles through differential centrifugation. Resuspension in Assay Buffer: Ensures that peroxisomes are in the proper environment for the catalase assay. Catalase Assay: This assay is the primary method to assess peroxisomal function. By measuring the degradation of H₂O₂, it allows for the determination of catalase activity in the isolated peroxisomal fraction. 19 BIOS 420L Cell Biology Concept Summary Exercise 1-10 Outcome: The main outcome of the exercise is the quantification of catalase activity in the peroxisomal fraction, which serves as evidence of successful isolation of functional peroxisomes. The results also provide a measure of the enzyme's efficiency in degrading hydrogen peroxide. STUDY THE CALCULATION SHEET FOR FINAL EXAM FOR EXERCISE 11-12: See separate files. 20