Western Blotting & Bradford Assay

Question 1

what is western blotting used for?

Answer 1

detects & quantifies specific proteins in a sample (for studying protein expression, cellular processes like apoptosis…)

Question 2

why is a lysis buffer used when isolating proteins from a whole cell extract for a Western Blot? what does a lysis buffer contain?

Answer 2

lysis buffer - breaks opens cells and releases proteins

contains:
- detergents (e.g. Triton X-100 = breaks down cell membranes, releases proteins)
- protease inhibitors = prevents protein degradation

Question 3

what is the importance in using PBS when harvesting cells? how would you harvest cells when trying to isolate protein from them?

Answer 3

harvest cells using mechanical scrapping - doesn’t damage/affect surface proteins like enzymatic scrapping

PBS - maintains cell integrity, keeps environment isotonic & stable for proteins

Question 4

how would you isolate proteins from a whole cell extract treated with a drug for a Western Blot? include reagents (6 marks)

Answer 4

1. seed cells - form a monolayer attached to each other
2. treat with drugs
3. collect/harvest cells with mechanical scrapping (to preserve surface proteins/condition of proteins) - use PBS to maintain cell & protein integrity
4. lyse cells - use lysis buffer to break open membranes and release proteins
5. prepare protein extract with vortex & incubation

Question 5

what is a Bradford assay used for?

Answer 5

quantifies total protein concentration in a sample - used to ensure equal amounts of protein are loaded during a western blot

Question 6

what dye does the Bradford reagent contain?

Answer 6

Coomassie Blue dye

Question 7

what is BSA? why is it used in the Bradford Assay?

Answer 7

BSA - standard protein used to generate a standard curve in the Bradford assay
- known concs establish relationship between absorbance & protein conc = used later to calculate unknown protein concs in samples (from a Western Blot)

Question 8

Role of NaCl in the Bradford Assay?

Answer 8

NaCl dilutes BSA solutions in the standard curve - maintains isotonic conditions, ensures accurate dilutions

Question 9

What is the Bradford Reagent and how does it work?

Answer 9

Bradford reagent - contains Coomassie Blue dye
- dye binds to proteins
- dye changes colour from brown to blue
- intensity of the blue colour measured at absorbance 595nm is directly proportional to the protein concentration

Question 10

How is protein concentration determined in the Bradford Assay?

Answer 10

• standard curve is created using known BSA concentrations and their corresponding absorbance at 595 nm
• absorbance values of the sample proteins measured
• values are compared to the standard curve to determine protein conc. of each unknown sample

Question 11

what is the importance of Bradford assays?

Answer 11

quantifies proteins to ensure correct sample loading for subsequent Western blotting or other assays

Question 12

what is an SDS-PAGE used for?

Answer 12

separates proteins based on their molecular weight by running them through a polyacrylamide gel under an electric field
- smaller proteins migrate further

Question 13

what is the importance of using an SDS buffer during SDS-PAGE?

Answer 13

SDS buffer denatures proteins (breaks down secondary & tertiary structures) - ensures they carry a negative charge for electrophoresis

Question 14

why are samples boiled before being run through SDS-PAGE?

Answer 14

denature proteins - break down structure so they migrate through the gel based on size alone

Question 15

what’s the importance of loading a biotinylated marker?

Answer 15

helps estimate the molecular weight of proteins by providing known reference sizes

Question 16

what is a biotinylated marker?

Answer 16

protein ladder labelled with biotin - allows visualization on blot using streptavidin-HRP chemiluminescent detection
- confirms semi-dry transfer success to membrane
- provides molecular weight reference

Question 17

what occurs during an SDS-PAGE?

Answer 17

1. gel prepared = pour resolving and stacking gels with appropriate concentrations
2. samples prepared = denature protein samples with SDS sample buffer & heat/boiling
3. load and run gel = load samples and markers, then run gel under an electric field (175V) to separate proteins by size

Question 18

what does the semi-dry transfer process in Western Blotting do? why is it done?

Answer 18

moves proteins from the gel onto the (nitrocellulose/PVDF) membrane
- (gel - separates proteins; membrane = holds them in place)

immobilises proteins so they can be probed with antibodies in Western blotting to detect specific proteins/proteins of interest

Question 19

why is blocking buffer added to the membrane?

Answer 19

blocking buffer (often has milk/BSA) - prevents non-specific binding of antibodies during detection

ensures the antibody binds only to the target protein

Question 20

what controls should be included in a chemiluminescence reaction?

Answer 20

1. positive control - known protein expected to be present = confirms antibody works
2. negative control - no primary antibody/ irrelevant antibody added = checks for non-specific binding
3. only secondary antibody added = no primary antibody - should rule out false positives through incorrect binding & signal

Question 21

What is Chemiluminescence in Western Blotting?

Answer 21

detection method - enzyme (usually HRP) reacts with a chemiluminescent substrate (e.g., Luminol)

produces light captured on X-ray film or a digital imager

Question 22

what reaction specifically produces the chemiluminescent signal in Western Blot?

Answer 22

enzyme-substrate reaction produces the signal - substrate must react with an enzyme to emit light (e.g. HRP & Luminol)

Question 23

what are the roles of primary & secondary antibodies during a chemiluminescence reaction?

Answer 23

primary antibody - binds specifically to target protein

secondary antibody - binds to the primary antibody and is conjugated to an enzyme (HRP/AP)

this system amplifies the signal - multiple secondary antibodies can bind to a single primary

Question 24

why is TBS-Tween used in washes in between the addition of antibodies in chemiluminescence?

Answer 24

TBST = Tris-buffered saline + Tween-20 (a detergent)
- washes away unbound antibodies
- Tween-20 prevents non-specific binding = reduces background signal
- maintains protein-antibody interactions during washes

Question 25

what's the importance of verifying loading with a housekeeping gene? how does this work?

Answer 25

purpose - confirms equal protein loading across all lanes (should show equal signal with HK gene); used as internal controls as they're constitutively expressed in most cells how: - probe membrane with antibody against a HK protein - band intensity should be consistent across lanes

Question 26

describe the process of a western blot involving a Bradford assay

Answer 26

1. sample cell lysis using a lysis buffer with detergents to release proteins & a protease inhibitor to prevent their degradation 2. protein quantification via a Bradford assay/ similar methods to ensure equal loading 3. mix protein samples with a sample buffer to denature proteins, ensure they sink into wells & add colour for tracking 4. 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 5. transfer proteins from gel to a (nitrocellulose) membrane using a transfer buffer 6. incubate membrane with a blocking buffer - prevents non-specific antibody binding 7. 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 8. enzyme-substrate reaction produces a signal (chemiluminescence) that indicates where the antibody has bound 9. visualise bands on the membrane to determine protein size & abundance

Question 27

why do we need to add a lysis buffer to sample cells for a western blot?

Answer 27

breaks open cells to release their protein contents - creates a whole-cell extract (population of all proteins in a sample)

Question 28

why are protease inhibitors added during cell lysis?

Answer 28

prevent protein degradation

Question 29

what is the purpose of a Bradford assay in a western blot workflow?

Answer 29

quantifies the amount of protein in the sample - ensures equal protein loading across all lanes

Question 30

what are the components of sample buffer in SDS-PAGE? what are they present for?

Answer 30

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

Question 31

why is glycerol an important component of sample buffer?

Answer 31

glycerol makes the sample dense so it sinks into the well

Question 32

what is the importance of beta-mercaptoethanol in sample buffer?

Answer 32

breaks disulphide bonds to denature proteins - allows proteins to unfold completely so they can be separated solely based off molecular weight

Question 33

what does SDS do in the sample buffer?

Answer 33

denatures proteins and gives them a uniform negative charge

Question 34

during gel electrophoresis, how are proteins separated?

Answer 34

by molecular weight - smaller proteins migrate further

Question 35

what is the purpose of transferring proteins from a gel to a membrane (e.g. nitrocellulose)?

Answer 35

makes the proteins accessible for antibody binding and detection

Question 36

describe the process of probing the membrane with antibodies

Answer 36

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

Question 37

how can we ensure that equal protein loading was maintained during the procedure?

Answer 37

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

Question 38

in Bradford assays, Coomassie dye binds to proteins and causes a colour change. what colour change is? what does the colour change indicate?

Answer 38

colour change from brown to blue - indicates presence of protein intensity of colour is proportional to protein concentration

Question 39

what is a limitation of the Bradford assay?

Answer 39

it doesn't identify specific proteins, only total protein concentrations

Question 40

(!!) What is the purpose of using a lysis buffer in western blotting? What are its key components?

Answer 40

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

Question 41

(!!) 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

Answer 41

C: triton X-100 - used in lysis buffers

Question 42

(!!) What role does SDS play in both the sample buffer and the polyacrylamide gel electrophoresis process?

Answer 42

denatures proteins by disrupting their secondary and tertiary structures & provides a uniform negative charge - ensures separation is solely off molecular weight

Question 43

(!!) Explain how proteins are separated by size during SDS-PAGE.

Answer 43

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

Question 44

(!!) You observe faint bands in all lanes of your western blot. What potential experimental errors could explain this?

Answer 44

insufficient protein loading poor transfer - pipetting small volumes is difficult insufficient substrate for detection

Question 45

(!!) Why is a housekeeping protein, such as GAPDH or actin, used in a western blot?

Answer 45

housekeeping genes serve as loading controls to confirm equal loading across all lanes - produce a consistent single band across all lanes

Question 46

(!!) What is chemiluminescence, and how is it used to detect proteins on a western blot?

Answer 46

chemiluminescence - a light-producing reaction between a substrate and an enzyme (e.g. horseradish peroxidase & substrate) to visualise protein bands on the membrane

Question 47

(!!) Why is a standard curve used in a Bradford assay?

Answer 47

allows the protein concentration of unknown samples to be determined by comparing their absorbance values to known standards

Question 48

(!!) You perform a Bradford assay and see no colour change in your protein sample. What might have gone wrong?

Answer 48

* absence of protein in sample * use of an incompatible buffer that interferes with the (Coomassie Brilliant Blue) dye

Question 49

(!!) At what wavelength is absorbance measured in a Bradford assay, and what does the absorbance value indicate?

Answer 49

absorbance at 595mm - value indicates the protein concentration based on the intensity of the blue colour

Question 50

(!!) Lane 1 in a western blot (containing the biotinylated marker) is completely absent. List possible reasons for this outcome.

Answer 50

- failure to load marker - improper marker preparation - incomplete transfer of proteins to the membrane

Question 51

(!!) 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?

Answer 51

- insufficient treatment to induce apoptosis - sample contamination - non-specific antibody binding (can be resolved by using a blocking buffer with BSA)

Question 52

(!!) What is the importance of BSA in western blots? What errors can it prevent?

Answer 52

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

Question 53

(!!) What might cause faint bands across all lanes of a western blot?

Answer 53

- insufficient protein loading - poor antibody binding - low substrate concs = insufficient chemiluminescence for visualisation

Question 54

(!!) If a western blot shows unexpected additional bands, what might this indicate about your sample preparation or antibodies?

Answer 54

- protein degradation (error in protease inhibitors) - non-specific antibody binding (flaw in blocking buffer/BSA)

Question 55

(!!) Compare the roles of detergents in lysis buffer versus sample buffer.

Answer 55

lysis buffer - detergents disrupt cell membranes to release proteins sample buffer- detergents denature proteins and coat them with a uniform negative charge

Question 56

(!!) TRUE/FALSE - the Bradford assay can differentiate between specific and non-specific proteins in a sample

Answer 56

FALSE

Question 57

outline key steps in performing a Western blot

Answer 57

Extract proteins and quantify using a Bradford assay to ensure equal loading. Run SDS-PAGE to separate proteins by size; transfer to a PVDF or nitrocellulose membrane. Block membrane, then detect the protein using primary and enzyme-linked secondary antibodies with chemiluminescent substrate.

Question 58

Why probe a housekeeping gene like GAPDH?

Answer 58

Housekeeping genes like GAPDH are consistently expressed - serve as a loading control to confirm equal protein amounts and validate the blotting protocol

Question 59

No signal on blot – two reasons + troubleshooting (2 marks)

Answer 59

Protein not present or poorly extracted → Troubleshoot by verifying lysis protocol and repeating extraction. Antibody issue or protein degraded → Use validated antibodies and include protease inhibitors during lysis.

Question 60

Role of primary & secondary antibodies in chemiluminescence detection (2 marks)

Answer 60

primary antibodies bind the target protein. secondary antibodies bind the primary and are conjugated to enzymes (e.g., HRP or AP). Upon substrate addition, a chemiluminescent signal is produced, reflecting protein levels.