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What does SDS PAGE stand for
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
What is the role of acrylamide in gel formation
Acrylamide is a monomer that polymerizes to form the gel matrix
What is the primary use of SDS PAGE
To separate proteins by their molecular weight
What happens to glycine zwitterions at higher pH in the stacking gel
They get deprotonated and stop contributing to the stacking effect
How does the stacking gel differ from the separating gel in SDS PAGE
The stacking gel has larger pores and a lower pH
How does SDS PAGE achieve protein separation
By using a gel matrix that allows proteins to migrate based on size
How is pore size related to acrylamide concentration
Higher acrylamide concentration results in smaller pore size
Why is pore size important in SDS PAGE
Pore size affects the migration rate of proteins through the gel
What molecules are necessary to make the gel for SDS PAGE
Acrylamide and bisacrylamide
What is the stacking effect in SDS PAGE
It concentrates proteins into narrow bands before separation
What would happen if a gel run was conducted without the stacking effect
Proteins would not be concentrated and would appear as broad bands
Under what scenarios might the stacking effect not work
If the pH of the stacking gel is not optimal for glycine
What is the relationship between protein size and the amount of SDS used
1 negative charge per 2 amino acids
How does boiling affect protein conformation in SDS PAGE
It destroys the 3D conformation but not the primary structure
What is the purpose of adding bromophenol blue to the SDS PAGE mix
To monitor the progress of the electrophoresis run
What is the gel matrix made of in SDS PAGE
Polyacrylamide
How is polyacrylamide formed for the gel
Through radical-induced polymerization of acrylamide and bisacrylamide
What is the effect of acrylamide concentration on pore size
Higher acrylamide concentration leads to smaller pores
What is Ohm's law in the context of SDS PAGE
Voltage is equal to current times resistance (V = I x R)
What is the significance of glycine's isoelectric point (pI) in SDS PAGE
It affects the behavior of glycine in the stacking gel
What ions are involved in the stacking effect
Glycine (trailing ion) and chloride ions (leading ion)
What factors affect the separation of proteins in the resolving gel
Percentage of acrylamide, ratio of acrylamide to bisacrylamide, and size differences
What happens to the stacking effect when glycine zwitterions are deprotonated
The stacking effect stops as they can no longer concentrate proteins
What is the function of sodium dodecyl sulfate (SDS) in SDS PAGE
To impart a negative charge to proteins
What is the role of TRIS buffer in SDS PAGE
To stabilize the pH of the gel
How can pore sizes be adjusted in SDS PAGE gels
By changing the concentration of acrylamide and bisacrylamide
What is the role of the resolving gel in SDS PAGE
To separate proteins based on their size
What are reducing agents used for in SDS PAGE
To reduce disulfide bonds between cysteine residues
At what pH does the Coomassie dye become negatively charged and visually blue
At around pH 2.
What is the effect of adding ethanol to the transfer buffer during protein transfer
It helps strip proteins from SDS, aiding their binding to the nitrocellulose membrane.
How does Coomassie staining indicate that protein samples were normalized before loading
The background pattern of proteins should look similar in intensity between different samples.
What is the role of milk protein (casein) in the blocking step of immunoblotting
It inhibits non-specific protein-surface interactions by covering unoccupied spots on the membrane.
How does the use of fluorescent dye molecules in secondary antibodies enhance detection
It allows for the detection of small variations in protein amount and multiplexing of different proteins.
What is the purpose of testing for a housekeeping protein in each sample
To ensure the same amount of sample was loaded in each well.
What can Coomassie staining of an SDS PAGE test for
It can test if a protein is pure in solution or if other proteins are present.
Which amino acid side chains does Coomassie Brilliant Blue dye bind tightly to
Arginine side chains.
How do hydrophobic amino acids affect binding specificity and affinity
They decrease specificity but increase affinity.
What could uneven distribution of the protein of interest indicate
It may not be due to different conditions but could be due to uneven loading.
What happens to the Coomassie dye at a pH above 1
One nitrogen atom is deprotonated, making the molecule neutral in charge.
What is the absorption peak of the red form of Coomassie Brilliant Blue dye
470 nm
What does the dissociation constant measure in the context of protein binding
It measures the affinity between two binding partners.
What happens if the blocking step is forgotten in immunoblotting
Antibody molecules will stick to the free membrane, causing non-specific signals.
Why is a secondary antibody used in immunoblotting
To increase sensitivity and reduce costs by using one batch for multiple primary antibodies.
What is the purpose of washing the membrane after adding the primary antibody
To remove non-specifically binding and non-bound primary antibodies.
What is the purpose of Coomassie Brilliant Blue dye in gel electrophoresis
It binds to proteins and reveals protein bands after washing out unbound dye.
What is the purpose of ladder proteins in gel electrophoresis
To determine the molecular weight of the protein of interest.
What must the secondary antibody be attached to before use
It must be covalently attached to either an enzyme or a fluorescent dye.
What is the visual result of the reaction involving alkaline phosphatase and nitroblue tetrazolium
A colorful precipitate appears on the membrane where the protein of interest is located.
What is necessary for multiplexing in fluorescent detection
The two primary antibodies must be raised in different animal species.
What are housekeeping genes responsible for
They encode proteins needed under almost all conditions at similar levels.
What is the role of hydrophobic pockets in proteins regarding Coomassie dye binding
They enhance the binding to Coomassie Brilliant Blue dye.
What is required for the detection of a protein of interest using antibodies
A specific primary antibody and a secondary antibody that binds to the primary antibody.
What is the reaction that occurs in colorimetric detection using alkaline phosphatase
It catalyzes the dephosphorylation of 5-Bromo-4-chloreo-3-indolyl phosphate to form a purple precipitate.
What are the three visualization methods used in immunoblotting
Colorimetric detection: Produces a colored precipitate where the enzyme is present. Chemiluminescent detection: Emits light upon decay of a reaction product. Fluorescent detection: Emits light when excited by a specific wavelength.
What is the function of alkaline phosphatase in the detection process
It removes the phosphate group from nitroblue tetrazolium, forming a colorful precipitate.
How can protein structures be visualized using software
By entering the PDB code into visualization software like pymol or Cn3D.
What is the reaction involved in chemiluminescent detection using horseradish peroxidase
It catalyzes the reaction of H2O2 and luminol to emit light at 425 nm.
What is a key advantage of second generation biofuels
No competition with food production
What is a trial overexpression and why is it advised
A trial overexpression helps determine optimal growth conditions for producing the POI
What are the different phases of a microbial growth curve
Lag phase: adaptation to new conditions. Log phase: rapid growth and division. Stationary phase: high cell density leads to metabolism shutdown
What is the purpose of running SDS-PAGE
To separate proteins by molecular weights
What pH conditions are needed for Coomassie staining
Anything above 2
What is the effect of reducing agents on antibody molecules in SDS PAGE
They allow separation of monomers from multimers
What is the charge of trailing ions in the buffer for them to enter the gel
They must be negatively charged
What does a primary antibody bind to
It binds specifically to the target protein
What are the options for visualizing proteins on a membrane
Colorimetric, chemiluminescent, and fluorescent detection methods
How does immunoblotting visualize the target protein
By using a secondary antibody conjugated to an enzyme or dye
What does the variable region of an antibody do
It binds to the epitope of the antigen molecule
Why is it not advisable to use the Bradford assay at pH 7
There won't be a color change because CBB is already in blue form
What molecule is measured in the spectrophotometer during the enzyme reaction
The amount of NAD+ reduced to NADH
What is the significance of the dilution factor in enzyme assays
It allows for accurate measurement of enzyme activity
Why is differential centrifugation particularly useful for eukaryotic cells
Because eukaryotic cells contain compartments that can be separated.
When would you use a monoclonal antibody
When you need to find a specific version of a protein recognized by one antibody.
What is the purpose of using various control samples in experiments
To ensure the validity and reliability of experimental results.
What class of enzymes are alcohol dehydrogenases (ADH)
ADHs are oxidoreductases that catalyze oxidation-reduction reactions.
Why should gloves not be worn outside of the lab
To prevent contamination and maintain a safe environment.
What are the advantages of biofuels compared to fossil fuels
Biofuels are renewable and do not add to atmospheric CO2 levels.
What is first generation biofuel made from
Edible feedstock, such as corn.
How can modern biotechnology be applied to produce biofuel molecules
By using microbes as factories that produce biofuel molecules through overexpressing enzymatic pathways.
Why is protein overexpression useful
It allows for the production of high amounts of the protein of interest for easier purification and analysis.
What is a trial overexpression
A method to determine optimal growth conditions for producing the protein of interest.
What parameters are typically tested for optimal overexpression conditions
Temperature, IPTG concentration, and duration of induction.
What components are needed on the plasmid for protein expression
Promoter, origin of replication, operator, lacI gene, ribosome binding sequence, selectable marker, His6 tag, and gene of interest.
What is the function of the T7 promoter on the plasmid DNA
It serves as a recognition site for T7 RNA polymerase for transcription of the gene of interest.
How do you transfer experimental data of product vs. time to the Michaelis-Menten graph
By plotting the linear part of the slope for each substrate concentration
What issue arises from using a first order enzyme assay for measuring enzyme concentration
You would underestimate the enzyme concentration due to limited substrate
Are all data points on the Michaelis-Menten graph initial rates
Yes, all data points represent initial rates
How do you calculate the original molarity of the enzyme from the absorbance change
Using the formula <latex>\Delta A = \varepsilon \cdot l \cdot \Delta C</latex> and adjusting for dilution
What should you review before entering a biochemistry lab
Familiarize yourself with the safe procedure and hazards of the chemicals.
What personal protective equipment (PPE) should be worn in the lab
100% cotton flame-retardant lab coat, long pants, closed-toed shoes, eye protection, and lab gloves.
What should you do in case of an accident in the lab
Call 911 from the lab phone.
What is the purpose of setting up hazardous waste containers before a lab experiment
To ensure safe disposal of hazardous materials generated during the experiment.
What is the function of the eyewash station in the lab
To rinse eyes for at least 15 minutes in case of chemical exposure.
What is a disadvantage of first generation biofuels
They compete with food production, impacting prices of staple food.
What is second generation biofuel made from
Plant waste material.
What is a disadvantage of second generation biofuels
They are much harder to digest due to biomass in cell walls.
What is third generation biofuel derived from
Algal cells.
What is an advantage of third generation biofuels
They can be engineered to produce biofuel molecules directly and do not compete with food production.
How is cell density measured
By measuring absorbance at 600 nm (OD600).
What happens if you induce a culture in the lag phase
Cells are preparing for growth, and forcing them to produce foreign proteins can inhibit their growth.
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