Western Blotting & Bradford Assay Flashcards
what is western blotting used for?
detects & quantifies specific proteins in a sample (for studying protein expression, cellular processes like apoptosis…)
what is a Bradford assay used for?
quantifies total protein concentration in a sample - used to ensure equal amounts of protein are loaded during a western blot
what is an SDS-PAGE used for?
separates proteins based on their molecular weight - protein samples are run through a polyacrylamide gel under an electric field, where smaller proteins migrate further
describe the process of a western blot
- sample cell lysis using a lysis buffer with detergents to release proteins & a protease inhibitor to prevent their degradation
- protein quantification via a Bradford assay/ similar methods to ensure equal loading
- mix protein samples with a sample buffer to denature proteins, ensure they sink into wells & add colour for tracking
- load equal amounts of proteins into wells of a polyacrylamide gel & run the gel by applying an electric current - separates proteins by molecular weight; smaller proteins migrate further
- transfer proteins from gel to a (nitrocellulose) membrane using a transfer buffer
- incubate membrane with a blocking buffer - prevents non-specific antibody binding
- add antibodies - a primary antibody specific to the target protein, a secondary enzyme-conjugated antibody specific to the primary antibody & a substrate for the enzyme
- enzyme-substrate reaction produces a signal (chemiluminescence) that indicates where the antibody has bound
- visualise bands on the membrane to determine protein size & abundance
why do we need to add a lysis buffer to sample cells for a western blot?
breaks open cells to release their protein contents - creates a whole-cell extract (population of all proteins in a sample)
why are protease inhibitors added during cell lysis?
prevent protein degradation
what is the purpose of a Bradford assay in a western blot workflow?
quantifies the amount of protein in the sample - ensures equal protein loading across all lanes
what are the components of sample buffer? what are they present for?
bromophenol blue - a dye to visualise loading
glycerol - makes the sample dense so it sinks into the well
beta-mercaptoethanol - breaks disulphide bonds to denature proteins
detergent (SDS) - denatures proteins & gives them a uniform negative charge
why is glycerol an important component of sample buffer?
glycerol makes the sample dense so it sinks into the well
what is the importance of beta-mercaptoethanol in sample buffer?
breaks disulphide bonds to denature proteins - allows proteins to unfold completely so they can be separated solely based off molecular weight
what does SDS do in the sample buffer?
denatures proteins and gives them a uniform negative charge
during gel electrophoresis, how are proteins separated?
by molecular weight - smaller proteins migrate further
what is the purpose of transferring proteins from a gel to a membrane (e.g. nitrocellulose)?
makes the proteins accessible for antibody binding and detection
describe the process of probing the membrane with antibodies
add a primary antibody specific to the target protein
add a secondary enzyme-conjugated antibody (e.g. conjugated with horseradish peroxidase) - this binds to the primary antibody
add a substrate which reacts with HRP enzyme = produces a chemiluminescence reaction, allows detection of protein bands on the membrane
how can we ensure that equal protein loading was maintained during the procedure?
use a housekeeping gene as a loading control
- probe the membrane with an antibody specific to the housekeeping protein
- verify equal protein loading through a consistent band across all lanes
in Bradford assays, Coomassie dye binds to proteins and causes a colour change. what colour change is? what does the colour change indicate?
colour change from brown to blue - indicates presence of protein
intensity of colour is proportional to protein concentration
what is a limitation of the Bradford assay?
it doesn’t identify specific proteins, only total protein concentrations
(!!) What is the purpose of using a lysis buffer in western blotting? What are its key components?
lysis buffer - breaks open sample cells to release their protein contents & obtain a whole-cell extract
components - detergent like SDS, Triton X-100, Tris-HCl & protein inhibitors added to prevent protein degradation
(!!) Which of the following is NOT a component of the sample buffer used in SDS-PAGE?
a) Bromophenol blue
b) SDS
c) Triton X-100
d) Glycerol
C: triton X-100 - used in lysis buffers
(!!) What role does SDS play in both the sample buffer and the polyacrylamide gel electrophoresis process?
denatures proteins by disrupting their secondary and tertiary structures & provides a uniform negative charge - ensures separation is solely off molecular weight
(!!) Explain how proteins are separated by size during SDS-PAGE.
equal loading of protein samples onto wells of a polyacrylamide gel using a pipette - then coat proteins with SDS & run an electric current through the gel to separate them via molecular weight
smaller proteins will migrate further down the gel matrix
(!!) You observe faint bands in all lanes of your western blot. What potential experimental errors could explain this?
insufficient protein loading
poor transfer - pipetting small volumes is difficult
insufficient substrate for detection
(!!) Why is a housekeeping protein, such as GAPDH or actin, used in a western blot?
housekeeping genes serve as loading controls to confirm equal loading across all lanes - produce a consistent single band across all lanes
(!!) What is chemiluminescence, and how is it used to detect proteins on a western blot?
chemiluminescence - a light-producing reaction between a substrate and an enzyme (e.g. horseradish peroxidase & substrate) to visualise protein bands on the membrane
(!!) Why is a standard curve used in a Bradford assay?
allows the protein concentration of unknown samples to be determined by comparing their absorbance values to known standards
(!!) You perform a Bradford assay and see no colour change in your protein sample. What might have gone wrong?
absence of protein in sample
use of an incompatible buffer that interferes with the (Coomassie Brilliant Blue) dye
(!!) At what wavelength is absorbance measured in a Bradford assay, and what does the absorbance value indicate?
absorbance at 595mm
- value indicates the protein concentration based on the intensity of the blue colour
(!!) Lane 1 in a western blot (containing the biotinylated marker) is completely absent. List possible reasons for this outcome.
- failure to load marker
- improper marker preparation
- incomplete transfer of proteins to the membrane
(!!) Lanes 2 (control) and 3 (treated) of your western blot show very similar band patterns, even though apoptosis was expected in lane 3. What could explain this?
- insufficient treatment to induce apoptosis
- sample contamination
- non-specific antibody binding (can be resolved by using a blocking buffer with BSA)
(!!) What is the importance of BSA in western blots? What errors can it prevent?
BSA - bovine serum albumin = used as a blocking buffer
prevents non-specific binding of antibodies to the membrane - ensures antibodies only bind to the target protein
(!!) What might cause faint bands across all lanes of a western blot?
- insufficient protein loading
- poor antibody binding
- low substrate concs = insufficient chemiluminescence for visualisation
(!!) If a western blot shows unexpected additional bands, what might this indicate about your sample preparation or antibodies?
- protein degradation (error in protease inhibitors)
- non-specific antibody binding (flaw in blocking buffer/BSA)
(!!) Compare the roles of detergents in lysis buffer versus sample buffer.
lysis buffer - detergents disrupt cell membranes to release proteins
sample buffer- detergents denature proteins and coat them with a uniform negative charge
(!!) TRUE/FALSE - the Bradford assay can differentiate between specific and non-specific proteins in a sample
FALSE
