I am performing western blot analysis to detect the protein expression changes in the brain tissues from several neurodegenerative diseases (e.g Alzheimer's, Parkinson's) as compared to normal control (age-matched). In your experience, which is a better loading control - GAPDH, ßActin or α/ßTubulin?
If you are aware of any articles that addresses above question, please do share.
Thank you for your feedback!!
Hi,
The journal of biological chemistry (JBC) declares in the editorial recommendations that "Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible. “House-keeping” proteins should not be used for normalization without evidence that manipulations do not affect expression.".
For more information the JBC indicates:
http://jbcmission.asbmb.org/collecting-and-presenting-data
Interestingly, Dr Oh informs that journals often openly state that of the western blotting normalization methods they prefer, total protein techniques are at the top of the list. In fact, since 2012 when it was first introduced, stain-free total normalization has appeared in over 800 publications (almost 500 of those appearing in 2017).
For more information
http://www.bioradiations.com/the-how-and-why-of-normalizing-your-western-blots/
I suggest as total protein quantification method the membrane staining with Ponceau S., Coomassie Blue or Amido black.
Best regards.
Mauricio
Firstly, since AD is associated with tau protein I felt tubulin protein is not the appropriate loading control. More examples for it you can find it here.
Front Cell Neurosci. 2015 Feb 23;9:24. doi: 10.3389/fncel.2015.00024.
So we used actin as loading control for our study. I would prefer actin or GAPDH, and not tubulin.
Loading controls are somewhat deceitful. I suggest you refer to the article below:
Rocha-Martins M, Njaine B, Silveira MS. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development. PLoS One. 2012;7(8):e43028.
doi:10.1371/journal.pone.0043028
Hi Mayur,
I think you should refer to MIQE guidelines and the use of more than 1 loading control for your samples. You may use Beta actin, GAPDH and two other constitutively expressing proteins as per general literature in your tissue type. Then you may determine using which reference gene, you are getting the minimum % coefficent of variance. You may nail down that particular reference gene for "your set of samples". I hope this helps.
Hi,
we have been working on Alzheimer's disease. we use Beta Actin and GAPDH as loading controls.
Hi
you could use b-actin
if your protein molecular weight is far away from it.
Hi,
The journal of biological chemistry (JBC) declares in the editorial recommendations that "Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible. “House-keeping” proteins should not be used for normalization without evidence that manipulations do not affect expression.".
For more information the JBC indicates:
http://jbcmission.asbmb.org/collecting-and-presenting-data
Interestingly, Dr Oh informs that journals often openly state that of the western blotting normalization methods they prefer, total protein techniques are at the top of the list. In fact, since 2012 when it was first introduced, stain-free total normalization has appeared in over 800 publications (almost 500 of those appearing in 2017).
For more information
http://www.bioradiations.com/the-how-and-why-of-normalizing-your-western-blots/
I suggest as total protein quantification method the membrane staining with Ponceau S., Coomassie Blue or Amido black.
Best regards.
Mauricio
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