Western Blot Sample Preparation

Written by westernblot

Sunday, 12 August 2007

Information on western blot protein sample preparation.

How to Prepare Protein Samples for Western Blot

Protein samples are usually prepared from either whole tissue or cell culture.

Preparing Protein from Tissue for Western Blotting

Solid organs and tissues must be broken down mechanically first using either a blender (for large organs or samples) or a homogenizer for smaller volumes.

Other methods are commonly used including French Press (which breaks cells down using pressure) and sonication.

During the procedures, cells are also broken open releasing proteases which can degrade your proteins. Therefore it is wise to keep everything on ice or colder, and also use protease inhibitors in your cell lysis or homogenization buffers.

western blot preparation

Detergents are used in the buffers during lysis to break down tissues and cells.

Salts are also added to help with breaking down the tissues and cells.

Buffers are used to keep the proteins at a constant pH during tissue breakdown and cell lysis. This allows proper solubilization of proteins and keeps them functioning properly.

Protease inhibitors such as PMSF and trazylol are always added to inhibit proteases which can degrade proteins in your sample.

A combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – can be used to separate different cell compartments and organelles.

Samples can then be broken down further using a dounce homogenizer or passing through a small gauge needle syringe in cell lysis buffer.

Cell Lysis Buffer for Protein Sample Preparation

Your choice of cell lysis buffer is very important for western blotting. If you require the analysis of enzyme activity you must use a non-denaturing cell lysis buffer. If you are looking to blot for phosphorylated proteins, you MUST keep your samples on ice and use relevant phosphatase inhibitors.

Tips for Western Blot Protein Sample Preparation

To get the best results, all the protein samples should be identical and in low ionic strength (low salt) buffers as salt affects gel electrophoresis and subsequent western blotting.
If you are comparing between samples and treatments, you must load the same amount of protein into each lane.
Make sure you determine the protein concentration using a Bradford assay, spectrophotometry as A280, or BCA. This will help you troubleshoot (weak or strong bands) and compare later.