Principles of Elisa and Western Blot techniques

Question 1

Understand the principles and applications of Western blotting and ELISA in biological research

Answer 1

WB:

• technique to detect and analyse proteins
• Applications:
• protein identification and quantification
• post-translational modification studies
• validation of protein expression levels in research or diagnostics
• mechanism: proteins are separated by size via SDS-PAGE, transferred to a membrane, and detected using specific antibodies

ELISA:

• uses antibody-antigen interactions for detection and quantification of specific molecules in solution
• applications : diagnostics, quantifying proteins, assessing immune responses
• mechanism: Enzyme-conjugated antibodies produce a measurable color change, fluorescence, upon reaction with a substrate
• Greater depth of colour = stronger binding

Question 2

Describe the process of Western blotting:

Answer 2

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

Question 3

Describe the process of ELISA:

Answer 3

Coating - antigen is immobilised on the surface of a well

Blocking - nonspecific sites are blocked to prevent false signals

Incubation with primary antibody - primary antibody binds to antigen

Addition of secondary antibody - the enzyme-linked secondary antibody binds to the primary antibody

Substrate addition to produce a colour change or light emission

Question 4

What are the different types of ELISA ?

Answer 4

Direct - single enzyme-linked antibody binds directly to the antigen

Indirect - primary antibody binds the antigen, followed by an enzyme-linked secondary antibody

Sandwich - capture antibody immobilised on a plate binds the antigen, followed by a detection antibody

Competitive - labeled antigen competes with the sample antigen for antibody binding

Question 5

Identify similarities and differences between Western blotting and ELISA

Answer 5

Similarities

• antibody based detection
• highly specific and high sensitivity allowing detection of low conc proteins
• both techniques use blocking agents
• used for protein analysis and quantification
• Each requires signal amplification

Differences:

Separation:
- WB = SDS-PAGE for protein separation
- ELISA = No separation; antigen in solution

Detection target:
- WB = specific protein bands
- ELISA = quantifies antigen concentration

Time
- WB longer
- ELISA faster for high-throughput testing

Output:
- WB band intensity on a membrane
- ELISA absorbance or fluorescence in wells

Question 6

Discuss potential challenges and troubleshooting strategies for WB and ELISA:

Answer 6

WB:

Weak bands - check transfer, antibody concentration
Nonspecific binding - optimise blocking

ELISA:

Low signal - optimise concentrations
High background - increase washing
Uneven results across sample replicates - ensure even antigen coating and use consistent incubation
times

Question 7

How is protein transfer confirmed in WB ?

Answer 7

Ponceau S stain used to visualise proteins on membrane

reversible stain with poor sensitivity

Ponceau S is easily removed with water and is
regarded as a “gentle” treatment that does not
interfere with subsequent immunological detection
steps