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Ten top tips for great western blots

Our Scientific Support team have put together what they think are ten top tips for great western blots.

1. Generally, it is better to incubate overnight at 4°C with the primary antibody

4°C overnight will usually provide more efficient and specific staining. However, many antibodies will also work very well with one or two hours incubation at room temperature.

2. Optimize the antibody concentration

Check the datasheet for a guideline starting concentration.

Background – reduce the concentration of antibody

No signal – increase the concentration of antibody

3. Use the appropriate gel percentage for the size of protein you are detecting

Protein size (kDa) Gel percentage (%)
4-40 20
12-45 15
10-70 12.5
15-100 10
25-200 8

4. Load the appropriate amount of sample

Cell lysates, membrane and nuclear lysates: load 20 to 30 μg of total protein per well.

Purified protein (recombinant or endogenous): load 10 to 100 ng of protein.

  • Reduce this amount for highly expressed proteins
  • Increase this amount for low expressed proteins

5. Use reducing and denaturing conditions unless stated on the antibody datasheet

Abcam antibodies have been tested under reducing and denaturing conditions only and we recommend using these conditions unless otherwise stated on the datasheet.

6. The blocking agent used can make a difference to results

Antibodies can be sensitive to blocking agents and we recommend reviewing the datasheet to see if there is any data to indicate the antibody works better with either BSA or milk. Generally, BSA will give clearer results as it contains fewer proteins for the antibody to cross react with. However, this is not always the case and some antibodies will work better with milk as it contains a greater variety of blocking proteins.

7. Don't let the membrane dry out at any time

This can affect antibody binding and give a 'grey' or 'black' blot.

8. Check the transfer to the membrane and quality of the sample using a loading control

View our list of loading controls.

9. Include a suitable positive control to assess how well the procedure and reagents are working.

There may be information regarding suggested positive controls on the datasheet. If not, try searching on protein database sites and PubMed for information on the protein expression.

View our list of selected controls for use in western blot.

10. Check the size of the band obtained using molecular weight markers

Remember, migration of the protein through the gel can be affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:

  1. Post-translational modification
  2. Post-translation cleavage
  3. Splice variants and isoforms
  4. Relative charge
  5. Multimers

Molecular weight markers ab48854 and ab41746.

More western blot resources from Abcam:

Please contact us for any other technical related questions.

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