Our Scientific Support team have put together what they think are ten top tips for great western blots.
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.
Check the datasheet for a guideline starting concentration.
Background – reduce the concentration of antibody
No signal – increase the concentration of antibody
|Protein size (kDa)||Gel percentage (%)|
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.
Abcam antibodies have been tested under reducing and denaturing conditions only and we recommend using these conditions unless otherwise stated on the datasheet.
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.
This can affect antibody binding and give a 'grey' or 'black' blot.
View our list of loading controls.
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.
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:
Please contact us for any other technical related questions.