The Protein Man's Blog | A Discussion of Protein Research

What's best? Housekeeping genes or total protein for Western blot loading controls

Posted by The Protein Man on Jun 29, 2015 2:34:00 PM
The Protein Man

Housekeeping_gene_Loading_ControlsWestern blotting is a very popular and sensitive protein detection system that is routinely used for multiple areas of protein research.  The sensitivity of this detection system continues to improve with increasingly sensitive enhanced chemiluminescence systems and the increasing popularity of IR (infra-red) fluorophores and IR imaging systems:

Are Housekeeping Genes A Suitable Control?

The advances in IR fluorophores and IR imaging systems do not negate the use of proper controls to account for technical errors and allow for normalization and correct processing of data. Loading controls routinely rely on expression levels of “housekeeping genes” to correct for differences in protein concentration or errors in loading. Routinely used housekeeping genes include β-actin, β-tubulin or GAPDH due to their high and ubiquitous expression across cell, tissues and disease/injury types. Although popular as controls, their validity as controls has been called into question for use in:

Cultured cells:

Tissue types:

Disease/injury state:

For researchers working on secreted proteins, such as cerebral spinal fluid (CSF), the above housekeeping genes are not secreted and therefore their validity as loading controls for biological fluids can likewise be questioned (Collins et al (2015)).

 Is Total Protein Staining A Suitable Alternative?

 Total protein stains are used to stain membranes after protein transfer and offer the following advantages over housekeeping genes as loading controls:

  • Faster: Majority of membrane stains stain within minutes, whereas blocking and antibody incubation steps for housekeeping genes can take hours,
  • Affordable: Compared to the cost of primary and labeled secondary antibodies, membrane stains are very affordable.  An additional cost saving is the quality of the transfer can be rapidly assessed before expensive antibodies and time are wasted on poor blots.
  • Reversible: Membrane stains are reversible and do not interfere with antibody labeling and imaging.
  • Save time:  The use of a membrane stain allows for increased sample throughput as a flurophore channel can be used for proteins of interest as opposed to housekeeping genes.

Total Protein Staining is A Reliable Control:

So it appears that total protein stains offer significant advantages over housekeeping genes as loading controls for Western blotting.

One major question still remains and has arisen due to the new IR imaging systems:

Are Total Protein Stains Compatible With IR Imaging Systems?

Click here to find out...

 

HB-Western-Blotting-HP   Antibody Production Handbook   HB-Sample-Preparation-HP

 

Topics: Western Blotting, Protein Detection

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