Western Blot Loading Controls

Loading Controls

Generate publication quality Western blots

Crucial Controls and Tips for IHC Experiments - Optimize your experiments

Loading Control Antibodies - Generate publication quality Western blots

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A Western blot provides a readout of differences in protein expression levels e.g. changes in protein levels over a certain time period, differences in knock-out versus wild type cell lines and altered expression in patient samples. However, in order to draw any conclusions from a Western blot one has to be sure that the observed differences are only due to altered protein expression levels rather than gel loading or protein transfer errors. For this purpose Western blot loading controls are used.

Sample type  Loading contol Molecular weight (kDa)*
Whole cell/cytoplasmic Alpha actin 43
Alpha tubulin 55
Beta actin 43
Beta tubulin 55
Cyclophilin A  18
GAPDH 37
Vinculin 116
Mitochondrial Cytochrome c oxidase 16
Hsp60 60
VDCA1/porin 31
Nuclear Histone H1/H3  
  Lamin B1 66
  PCNA 29
  TATA binding protein TBP 38
Serum Transferrin 77
*The exact molecular weight might vary from species to species

What are loading controls?

  • An antibody that detects a highly expressed protein in a certain cell or sample type.
  • Encoded by 'housekeeping genes', these proteins are selected due to their crucial involvement in maintaining cellular functions.
  • Tools for normalizing gel loading differences and Western blot quantification (by software programs such as ImageJ).

Why are loading controls crucial?

  • Loading controls are particularly necessary when signal levels of modified proteins are compared between samples.
  • They ensure you have loaded and transferred equal amounts of protein across all wells of your Western blot.

Choosing and using the right loading controls

  1. It is important to make sure your protein of interest and your loading control have different molecular weights.
  2. Ensure your loading control is highly expressed in your sample (see table) and its level remains unchanged throughout an experiment.
  3. Probing with a loading control can be carried out along with the target antibody (e.g. cutting the membrane) or later after the blot has been stripped of previously bound antibodies. 

Did you know?

  • For high molecular weight (over 100 kDa) proteins we recommend Vinculin as a loading control.
  • GAPDH is listed as a Top 20 housekeeping gene with the highest and most consistent average expression (She et al., 2009). Therefore GAPDH, together with beta actin and tubulin is one of the most commonly used loading controls.
  • We offer alpha tubulin and GAPDH DyLight® 680, DyLight® 750 and DyLight® 800 conjugated antibodies which are perfect as loading controls in fluorescent Western blotting experiments.
  • Both these products are also available directly conjugated to HRP, a quick alternative to using a secondary antibody.

References

She, X, et al., (2009) Definition, conservation and epigenetics of housekeeping and tissue-enriched genes.
BMC Genomics 10:269.

More tips and tricks about how to improve your experiments can be found here:

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Western blot guide

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What is Western blotting?

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Epitope tag antibodies

Interested in learning more about controlling your experiments:

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Alexa Fluor® is a trademark of Molecular Probes Inc, OR, USA.