Western Blot Protocol

Written by westernblot

Sunday, 26 August 2007

A very detailed western blot protocol for molecular biology researchers conducting western blotting in the laboratory.

Detailed Western Blot Protocol

This is the most detailed and comprehensive western blot protocol ever prepared. Please contribute to it so we can all benefit.

There is a commenting ability below, please add comments or suggestions there and we will incorporate those into the next revision!

Western Blot Protocol Steps

Sample Preparation

Gel Preparation

Running Gel

Transfer of Gel

- Controls

- Western Blot

- Readout

- Analysis and Interpretation

Western Blot Sample Preparation Protocol

Before you run a western blot, analyze what cell lines or tissues you are using. This will affect how you prepare your samples.

Also take precautions to prevent degradation of proteins by proteases. If you are analyzing phospho-proteins or phosphorylation, you need to inhibit phosphatases.

Put Samples, Cell Lines in Flasks or Tissues on Ice Bucket (see Lysis notes).
(for non-adherent cell lines, centrifuge gently and pellet).
Wash (pellet - for adherent cells) in PBS Solution.
Remove PBS Solution.
Add Cell lysis buffer (see Lysis Buffer Recipe).
Scrape adherent cells off flasks using scraper.
Re-suspend non-adherent pellet in lysis buffer.
Pass samples through 25 gauge needle syringe and/or Homogenize samples with dounce homogenizer.
Store and aliquot samples at -80°C freezer
Using an aliquot of the sample, analyze protein concentration.
To an aliquot (10-30 µg of the prepared cell lysate) add SDS-PAGE sample buffer (amount added depends on type of gel apparatus run and well size - mini vs larger diameter wells). This is usually 10ul to 40ul and also depends on the protein concentration to load.
Boil Samples 90°C-100°C heating block.
Samples are ready to load or store in fridge (-20°C for longer storage ) for quick usage.

Lysis Buffer Recipe for Western Blotting

A recipe for a cell lysis buffer.

10 mM Tris, pH=7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1 mM NaF
20 mM Na4P2O7
2 mM Na3VO4
0.1% SDS
0.5% sodium deoxycholate
1% Triton-X 100
10% glycerol
60 µg/mL aprotinin
10 µg/mL leupeptin

Just before using add:

1 mM PMSF (made from a 0.3 M stock in DMSO) - OR 1:1000 dilution 100 mM PMSF in isopropanol OR 1 mM AEBSF (water soluble version of PMSF)
1 µg/mL pepstatin (alternatively, protease inhibitor cocktail may be used)

See Notes below for western blot details.

Prepare Samples for Western Blot:

Lysis Notes:

Remove extracellular proteins from media for non-adherent cell lines (including T-cell lines or peripheral blood cells) or addherent cell lines (including fibroblast cells etc.)
Non-adherent cell lines can be pelleted with centrifugation and then wash pellet with PBS
Wash adherent cell lines with PBS in the flask
This greatly reduces background of proteins such as albumin

Protein Preparation for Western Blotting

Prevent protease degradation of proteins by:

Keep Samples on Ice at all times!
Use Protease inhibitors!

Analyzing Phosphorylation or Phospho-Proteins

If you are analyzing Phosphorylation or Phospho-Proteins, you need phosphatase inhibitors in your lysis buffer.

NOTE: A good tip for difficult western blot detections of phospho-protein analysis is to lyse DIRECTLY in Laemmeli SDS-PAGE loading buffer (100ul or less). Load the entire amount directly on a gel.

Detergent - Lysing Cells For Western Blot

Detergent to break up membranes of cells to release proteins

SDS and RIPA are detergents that are used to solubilize proteins for later analysis using western blotting. This is required as many proteins are either inside the cell or located inside cell membranes, so we need to release these proteins to be able to immunoblot for them.

Homogenization and Gauge Needle for Lysing Western Blot Samples

For efficient lysis you need to use a dounce homogenizer and/or pass samples through a 25 gauge needle syringe to disrupt the membranes. Sometimes this step is not needed.