Western Blot Flashcards
Why is it important to block the membrane before adding the primary antibody?
Blocking prevents non-specific binding of the primary and secondary antibodies to the membrane
This reduces background noise
Typically done using bovine serum albumin (BSA) or non-fat milk to cover non-specific protein-binding sites
What is the role of the SDS-PAGE step in Western blotting?
SDS-PAGE separates proteins based on their molecular weight by denaturing them and providing a uniform negative charge
Ensures proteins migrate according to size allowing for accurate identification
What factors could lead to non-specific bands ?
Insufficient blocking - incomplete blocking allows antibodies to bind non-
specifically to the membrane
High antibody concentration - excess primary/ secondary antibody can lead to off target binding
Cross reactivity - primary antibody may recognise similar proteins, leads to additional bands
Dirty/ contaminated equipment - contaminated buffers, membranes, or pipettes can introduce unwanted signals
Overexposure of detection system - Hugh exposure times can enhance background noise and cause misleading bands
Compare and contrast Western blotting with other protein detection methods such as ELISA or immunohistochemistry:
Western blotting detects proteins based on size and antibody specificity after separation by gel electrophoresis
ELISA is a plate-based assay that quantifies proteins in solution using antibody antigen interactions. It is more sensitive but does not provide molecular weight information
Immunohistochemistry (IHC) localizes proteins in tissue sections using labelled
antibodies, allowing spatial visualisation but lacking precise quantification
A researcher wants to quantify the amount of a specific protein using Western blot. What controls should they include, and how can band intensity be analysed ?
Controls: Include a loading control (e.g., β actin or GAPDH) to normalize protein amounts and a negative control to confirm specificity
Band intensity analysis: Use image analysis software (e.g., ImageJ) to quantify signal intensity, normalizing target protein bands to the loading control for accurate comparisons
What is the significance of including a loading control (e.g., actin or GAPDH) in a Western blot experiment?
Loading control is essential for ensuring equal protein loading across all lanes
Allows for normalisation of target protein levels
Accounts for variations in sample loading, transfer efficiency, and protein quantification
Ensures that observed differences in band intensity reflect actual biological changes rather than technical inconsistencies
Describe the process of a Western Blot:
Sample preparation - lyse cells and extract protein, denature proteins with SDS
Gel Electrophoresis - protein
sorting by size
Membrane Transfer - Transfer proteins from the gel onto a nitrocellulose or PVDF membrane
Blocking - prevent nonspecific antibody binding using a blocking agent
Primary Antibody Incubation - add an antibody specific to the target protein and incubate overnight
Secondary Antibody Incubation - add an enzyme-linked secondary antibody that binds the primary antibody
Western Blot Analysis - use a substrate that reacts with the enzyme to produce a visual signal
Describe the purpose of a Western Blot:
Western blotting is a widely used technique to detect and quantify specific proteins in a complex sample
Combines electrophoresis, membrane transfer, and antibody-based detection for precise identification of target proteins
Commonly used in biomedical research to:
Measure protein expression levels.
Detect post-translational modifications (e.g., phosphorylation).
Verify protein purification or recombinant protein expression.
Describe the interpretation of band patterns:
Compare sample bands to the molecular weight ladder to determine protein size
Band intensity can indicate relative protein abundance
Faint bands may suggest low protein expression or insufficient antibody binding
Multiple bands may indicate:
- post translational modifications
- protein degradation or cross reactivity with non-specific proteins
What factors could lead to weak signals ?
Low protein loading - insufficient sample leads to faint or undetectable bands
Inefficient protein transfer - poor membrane transfer from SDS-PAGE can reduce signal intensity
Low antibody concentration - using too little primary or secondary antibody weakens signal detection
Degraded target protein - protease activity in the sample can break down protein of interest
Poor ECL detection - weak or expired substrate can reduce signal strength
Describe some common troubleshooting errors and their causes and solutions:
Smeared bands:
- caused by overloaded protein sample
- solution is to reduce sample volume or conc
Non-specific bands:
- caused by cross reactivity
- Use a more specific antibody or increase blocking strength
Uneven bands:
- caused by bubbles trapped during transfer
- ensure even gel membrane contact
Excessive background:
- caused by incomplete washing
- increase blocking time + increase washing steps
Weak bands:
- cause is protein degradation
- add fresh protease inhibitors
No bands observed:
- low antibody concentration
-
Describe the controls used in a Western blot:
Positive controls - Confirm the primary antibody detects the target protein correctly
Negative controls - ensure antibody specificity by including samples known not to express the target protein
Loading controls - detect housekeeping proteins to confirm equal sample loading
Blank controls -
