How to chose right loading control for Western blot?

In biology there are no universal controls. One has to determine a priori what does not change in one's test conditions and use that as a control. The choice of appropriate house-keeping protein would depend upon the particular experiment, and it needs to be experimentally confirmed. House-keeping proteins cannot be used for normalization if there is a suspicion that experimental conditions are affecting their expression. Different stress conditions may for example affect levels of actin and tubulin, which will not make those proteins suitable as a loading control.

Protein used as a loading control and target protein are often not expressed in comparable levels, which leads to differences in linear detection range. This has to be taken into consideration and carefully optimized and adjusted by titration of protein load and taking into account that diferent exposure time may be necessary to record results (for chemiluminescent detection).

Other possible internal controls

• Total protein (express fmol target protein/µg total protein)
• Dry mass
• Total chlorophyll (express fmol target protein/µg chlorophyll)
• Cells (express fmol target protein/cell)
• Total nitrogen (express fmol target protein/fmol N)
• Experiment specific ratios of particular proteins (ex. fmol PsbA/fmol RbcL)

For short term experiments, Rubisco protein makes a good loading control as its turnover is slow. However, Rubisco level do vary with nutrient levels, particularly nitrogen and Rubisco protein may not always be the most optimal loading control.

Special notes

FtsZ can be used as a loading control in cyanobacteria.
The use of GAPC1/2 as a loading control should be supported with specific experimental data.
RbcL or RA (Rubisco activase) OEC223, AtpB (for Chlamydomonas reinhardtii, Rasala et al. 2013) or PsbO (OEE1) or PsbP (OEE2) for Chlamydomonas reinhardtii.
PsbO (OEE1) or PsbP (OEE2) are not so suitable for higher plants as accumulation of these proteins might drop to 12.5-25 % of the WT level in mutants defective for PSII core (Schult et al. 2007).
Tubulin alpha and gamma cannot be applied as a loading control for metal stress conditions. For more information check here.

Measureing protein concentration in each sample is crucial, to allow loading based on protein, not volume. There are reagents, which will allow to measure protein concentration in presence of detergents and other compounds of extraction and loading buffer.

How to chose right loading control for Western blot?

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