WESTERN BLOTTING

? Presented by
MAHESH KUMAR SIVASUBRAMANIAN
 INTRODUCTION
• 1979 - W. Neal Burnette – Seattle on the west coast

• Immunoblotting

• Detection and analysis of proteins

• Antibody: protein complex

 WORKFLOW
Sample preparation Gel electrophoresis Transfer

Antibody probing

Analysis Imaging Detection

 SAMPLE PREPARATION
• RIPA lysis buffer + Cocktail Inhibitors
• RIPA (Radio-Immunoprecipitation Assay) lysis buffer.

• Tris-HCl - Buffering agent - prevents protein denaturation

• NaCl (salt) - prevents non-specific protein aggregation

• NP-40 - non-ionic detergent to extract proteins

• Na-deoxycholate & SDS - ionic detergents to extract proteins

 SAMPLE PREPARATION
• Halt Protease and Phosphatase inhibitor (Single use Cocktail)

• Protease inhibitors - Amino peptidases, cysteine and serine proteases.

• Phosphatase inhibitors - Serine/threonine and protein tyrosine

phosphatases.

• EDTA & Pepstatin A (not included)

 SAMPLE PREPARATION
• Punched out mouse brain samples + RIPA lysis buffer in Eppendorf

• Ultrasonication (20-50KHz)
• The sound waves generate a region of low pressure, causing disruption of the
membranes of cells in the vicinity of the probe tip.

• Short bursts to avoid heat production. Keep in ice box.

• DNA release - highly viscous samples. To prevent add DNase

 SAMPLE PREPARATION
• Centrifugation @5000 rpm for 5mins – sufficient

• Supernatant has protein part

• This is used as sample for western blot

• For short time use, store it in -20 C & For long time use, store it in -80
C freezer
 SAMPLE PREPARATION
• Determine the conc. of protein to be loaded. Mix it with sample
loading buffer.
• Mix and boil for 5 mins.

WHY?
 SAMPLE PREPARATION
• Preparation of Sample loading buffer
• 950 µL 2X Laemmli sample buffer+ 50µL β- mercaptoethanol
• Cleave disulfide bonds b/w cysteine residues - Linear polypeptides

• Components of 2X Laemmli sample buffer

• Tris-HCl, pH 6.8
• Glycerol - Inc. the sample density and avoid mixing with running buffer
• SDS - Binds with protein (1.4g SDS with 1g protein)
• Bromophenol Blue - Monitor the migration through the gel
 GEL ELECTROPHORESIS
• Running buffer – Laemmli 10X Tris/Glycine/SDS buffer (100mL) +
distilled water (900mL)

• Fix the Precast gel in the inner chamber and fill the inner & outer
chamber with Running buffer.

• Set up the apparatus and run for 1hr 10mins @ 150V

• Proteins separate according to their MW

GEL ELECTROPHORESIS
 GEL ELECTROPHORESIS
Role of Glycine

pKa1= 2.3 pI= 5.95 pKa2= 9.6

 GEL ELECTROPHORESIS
(-)
pH 6.8

1- Cl (-)

2- Protein - SDS complex (-)

3- Glycine (N/-)
pH 8.8

(+)
GEL ELECTROPHORESIS

3 pH 6.8

1
GEL ELECTROPHORESIS

pH 8.8

1
 TRANSFER
• Transfer buffer
• 100mL Tris/ Glycine
• 200mL Methanol
• 1700mL Water
• Sponges
• Filter paper
• Transfer Membrane
• PVDF (Immobilon- P) - Polyvinylidene fluoride

• Soak the sponges and filter paper in transfer

buffer

• Soak the membrane in Methanol (3-5s) > water

(5minutes) > transfer buffer

• Transfer for 1hr @ 100V.

 TRANSFER
Why prewet PVDF
In Methanol?
• Very hydrophobic

• Short rinse in Methanol will "hydrate"

the membrane and allow improved
transfer and protein binding
 TRANSFER
• After transfer remove and disassemble the cassettes

• Place the gel in a container for Gel code blue staining

• Done to check the efficacy of transfer

• Place the blot into a box with TBST and soak for 5 mins
 ANTIBODY PROBING
• Incubate the blots for 3hrs in blocking buffer in cold room on rocker

• Avoid the non-specific binding of antibodies

• Add primary antibody to the blots and incubate it overnight in cold

room on rocker. Rinse the blots

• Add secondary antibody (with HRP) to the blots for 1hr in cold room
on rocker. Rinse the blots
ANTIBODY PROBING
 DETECTION
• Incubate the blots in ECL for 1 min

Luminol

(o) HRP

Emission of light
(protein quantity)
IMAGING