Western blot FAQ
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Frequently asked questions for western blotting.
Overview
Why are the band sizes different from expected?
How much sample should be loaded?
There are difficulties obtaining a band when detecting a recombinant protein. Why is this?
Do the samples need to be reduced and denatured?
Is there a difference between milk and BSA blocking?
Can I incubate the primary antibody at room temperature for one hour, instead of 4°C overnight?
Why are the band sizes different from expected?
Western blotting is a technique that separates proteins based on size. In general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:
- Post-translational modification - e.g. phosphorylation, glycosylation etc., which increases the size of the protein.
- Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases.
- Splice variants and isoforms - alternative splicing and isoforms may create different sized proteins produced from the same gene.
- Relative charge - the composition of amino acids (charged vs non-charged), and the formation of dimers and multimers, can affect how far the protein will travel through the gel. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands.
How much sample should be loaded?
The exact amount of sample to load may require some optimization, and will depend on your sample type. Our general recommendations are below:
- Cell lysates, membrane and nuclear lysates: 20 to 30 µg of total protein per well.
- Purified protein (recombinant or endogenous): 10 to 100 ng of protein.
If the bands appear large and diffuse (as in the image below), it is generally an indication that you've used too much sample. Try diluting the protein, and see if this gives clearer bands.
There are difficulties obtaining a band when detecting a recombinant protein. Why is this?
There are inherent difficulties with antibody detection of recombinant tagged proteins that need to be considered. We would recommend ensuring that the recombinant protein expressed in the sample includes the immunogen sequence for the antibody that you are using.
Also, if the recombinant protein is tagged, it is possible for the tag to prevent access of the antibody to the epitope. It is always better to place the tag at the C- or N- terminal end of the recombinant protein when possible, to reduce the chance of this occurring (rather than in the middle of the reading frame of the protein).
We would always recommend running an endogenous positive control when detecting recombinant proteins in western blotting.
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Do the samples need to be reduced and denatured?
We recommend reviewing the datasheet for further details. Abcam antibodies will have been tested under reduced and denatured conditions only and we recommend using reducing and denaturing conditions unless otherwise stated on the datasheet.
In some cases, they will also have been tested under non-reducing conditions and we will always specify on the datasheet when this is the case (see application notes section or Abreviews). The datasheet will always indicate if we have data to indicate the antibody will not work in one or other condition.
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Is there a difference between milk and BSA blocking?
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. When this is known, Abcam will always state this on the datasheet.
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 for more different types of proteins for blocking.
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Can I incubate the primary antibody at room temperature for one hour, instead of 4°C overnight?
The time of primary antibody incubation in western blotting will require optimization by the end user. Generally, 4°C overnight will give more efficient and specific staining. However, many antibodies will also work very well with one or two hours incubation at room temperature.
We recommend reviewing the individual datasheet for any data regarding suitable incubation times (see available Abreviews, publications and image data).
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