How to Prepare Samples for Western Blots

 

This guide provides all the information you need to prepare samples for Western Blots. Written by our PhD qualified expert antibody team, the guide includes protocols for protein extraction from tissues and cell culture, together with useful recipes for the buffers that you will need.

 

Contents

1. Protein Extraction from Tissues

2. Protein Extraction from Cell culture 2.1 RIPA Buffer Protocol 2.2 Laemmli Buffer Protocol

3. Recipes 3.1 RIPA Buffer 3.2 10x PBS 3.2 2x Laemmli sample buffer

 

1. Introduction

Proteins must be extracted and solubilised from any experimental sample before being used in a Western blot experiment. Lysis buffers are used to both break open cells and solubilise the proteins. Depending on the characteristics of the target protein and subcellular localisation the buffer conditions may require some optimisation. The protocols detailed here are intended to provide a starting point which can then be modified after experience. The protocol also differs between whether your samples derive from dissected tissues or cell culture.

 

2. Protein Extraction from Tissues

Ensure that all your tools are set up and buffers are ready before commencing the dissection as avoiding delays will help avoid protein degradation.

 

1. Use clean tools to dissect the tissue on ice working quickly to prevent protease degradation of the sample proteins.
2. Once the tissue is removed then rinse briefly in ice-cold PBS before cutting into small chunks using scissors.
3. Transfer the tissue to a homogeniser and add RIPA buffer (with protease inhibitors)
   1. Add 50µl RIPA buffer per 1mg of tissue (50ml of RIPA buffer per 1g tissue). This is a good starting point but can be adjusted depending upon the expression of the target/s of interest.
4. Homogenise the tissue thoroughly on ice before leaving the sample for 30 minutes on ice.
   1. It can be helpful to occasionally vortex the sample during this phase.
5. Use a sonicator while keeping the sample on ice to shear genomic DNA.
   1. The power settings will need optimising dependent upon the machine specifications and the sample volume
   2. Take care not to heat up the sample or induce too many bubbles during sonication.
6. Centrifuge the sample at 10,000g_av for 20 minutes at 4°C to pellet any cellular debris.
7. Transfer the supernatant to fresh tubes and aliquot.
   1. Tip: Use one aliquot to estimate the protein concentration. Be aware that the detergent in the sample can complicate some protein assays.
8. Snap freeze samples in liquid nitrogen and store at -80°C

 

3. Protein Extraction from Cell culture

We have provided two protocols: a traditional method using RIPA buffer which allows protein quantification and a quick method using Laemmli loading buffer. For both protocols ensure that all your tools are set up and buffers are ready before starting as avoiding delays will help avoid protein degradation.

 

3.1 RIPA Buffer Protocol

This protocol is suitable for if proteins need to be quantified or used in purposes other than western blotting.

1. Remove the cell culture dish from the incubator, place on ice then wash the cells with ice-cold PBS
   1. Tip: This step may need to be omitted for poorly adherent cells
2. Add ice-cold RIPA buffer (with protease inhibitors) to the cells and then use a cold plastic cell scraper to remove adherent cells into the buffer.
   1. A good starting point is 1ml RIPA per 10⁷ cells however this will need optimising depending upon the expression of the target protein/s
3. Transfer the RIPA buffer suspension into an ice-cold microcentrifuge tube then agitate for 30 minutes at 4°C
4. Centrifuge at 16,000g_av for 20 minutes at 4°C to pellet any cellular debris.
5. Transfer the supernatant to fresh tubes and aliquot
   1. Tip: Use one aliquot to estimate the protein concentration. Be aware that the detergent in the sample can complicate some protein assays.
   2. Tip: If the supernatant is extremely gloopy then it can be beneficial to sonicate the sample to break up genomic DNA.
6. Snap freeze samples in liquid nitrogen and store at -80°C

 

3.2 Laemmli buffer protocol

This is a quick protocol suitable for experiments where just a quick check for expression is needed or where the total protein concentration isn’t important.

1. Remove the cells from the incubator and place on ice.
2. Add 1x Laemmli sample buffer to cells then use a plastic cell scraper to remove adherent cells
   1. The amount of sample buffer needed will depend upon many factors but we find that 200µl works well for a confluent 25mm coverslip.
3. Transfer lysate into fresh tubes and either use immediately in experiments or store at -20°C
   1. Tip: If the supernatant is extremely gloopy then it can be beneficial to sonicate the sample to break up genomic DNA. Additionally this can be due to high protein concentration therefore diluting the sample in more 1x Laemmli sample buffer can be beneficial.

 

4. Recipes

4.1 RIPA Buffer

Notes:

• Store at 4°C for no longer than a couple of weeks as the solution tends to go cloudy over time
• Add protease and phosphatase inhibitors (if necessary for your experiment) immediately before use. Please see our guide on protease and phosphatase inhibitors for more information.