Hi Sadia,

The controls you need depend on the experimental sample treatment.

Typical examples

Assessing protein levels of untreated samples

In the simplest western blot to assess protein levels of a particular gene, blot for that protein, plus a the protein product of any house keeping gene such as Gapdh, tubulin, actin, etc. as a loading control. Loading control tells you how that equivalent amounts of each sample was loaded and is very important for comaprative analysis and downstream densitometry if desired.

Typical example:

Assessing protein levels of siRNA knockdwon samples

You need to assess the knockdown of a gene, say giantin, with giantin siRNAs. Include a positive control siRNA say, lamin a/c siRNAs, and a negative control siRNA which doesn't target any gene (usually called scrambled siRNAs).

When performing the western blot, blot with:

1. anti-giantin to assess giantin knockdown

2. anti-lamin a/c to show that your knockdown reagents/protocol worked fine

3. anti-Gapdh, anti-tubulin or antibodies to any house keeping gene to show that you loading equivalent amounts of protein in each sample.

@ David , Thanks for sharing detailed answer . much appreciated :)

Regards

Hi Sadia, the reasons that you have given in your question are correct with regard to GAPDH and b-actin. However, you may want to try them out first before disregarding either GAPDH or b-actin as loading control because the mere fact that the experiment alters or may alter metabolism or the cytoskeleton does not automatically mean that either of these genes will be involved/ affected. E.g. For non-energy metabolic processes, GAPDH is less likely to be involved/ affected. Likewise, b-actin is just a component of the cytoskeleton and may not be the very gene that is affected by the disease/ treatment. I would try it out first before switching to vinculin or tubulin (all components of the cytoskeleton). GAPDH and b-actin Abs are very easy to come by and that is why I would try them out first.

Other considerations in choosing your loading control may include the size of your gene relative to the housekeeping gene: they should be close in size but distinctive enough to detect them apart. It should be abundantly expressed in the cells/ tissue and remain unaffected by the experimental procedures.

For more insight, please read: She, X, et al., (2009) Definition, conservation and epigenetics of housekeeping and tissue-enriched genes.

BMC Genomics 10:269.

@Lawrence Owusu: Thanks prof , its pretty helpful . :)

Best Regards

Hi Sadia! just to add another thing. I would not use GAPDH as cytosolic control. My friends who were doing western blot with cell fractions always see GAPDH in nuclear fraction, and then we find this, which explains why!

Article Nuclear translocation of glyceraldehyde-3-phosphate dehydrog...