Problem in choosing internal contol for Western blot - not actin, tubulin, GAPDH and VDAC (Jul/03/2006 )

Dear all, I am doing WB on leukemia cell lines comparing between proteins of pre-treatment of drug and post- treatment of drug.
By using 2D and MS, I found actin, tublin, GAPDH, VDAC were changed after treatment of drug,
So what loading contol can I use for the WB of total cell lysate?

Many thanks.

Possible choice:
Histone
PBGD
lamin
orange G
mitochondrial ATPsy6 (mATPsy6)

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hi
i would use lamin in first try, as a mitochondrial protein is less reprducible in whole cell extraction.
i don't know orange G and PBDG.
Histone should be ok too.

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have a look at this:

Abcam loading control suggestions

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To find a protein that is not changed in any situation is so difficult


1: Electrophoresis. 2006 May 10; [Epub ahead of print] Related Articles, Links


beta-Actin is not a reliable loading control in Western blot analysis.

Dittmer A, Dittmer J.

Klinik fur Gynakologie, Universitat Halle, Halle, Germany.

beta-Actin is often used as a loading control in Western blot analysis. We analyzed the ability of beta-actin-specific antibodies to recognize differences in protein loading. We found that, at higher total protein loads as required for the detection of low-abundance proteins, beta-actin-specific antibodies failed to distinguish differences in actin protein levels. Diluting the antibody working solution or changing the incubation time had little effect on this phenomenon. This shows that beta-Actin is not a reliable loading control in Western blot analysis. In general, it appeared that, at longer incubation times, antibodies seem to be less able to pick up differences in the level of its target protein.

PMID: 16688701 [PubMed - as supplied by publisher]

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Is this suitable to use a mitochondrial loading control for the whole cell lysate?

But in my case, the loading control for mitochondial---- VDAC also changed.

So I am quite afraid to use other mitochondrial loading control.


Can I use spiked protein as loading control?

For example, I add a protein maybe with tag during the protein extraction step, there is no way that the cell lysate contain this protein, since the extraction ratio for every protein are the same, so I use this spiked protein as a loading control.

Can it be a good idea?

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true - also read this one:

Proteomics 2005, 5, 566–571
Housekeeping proteins:
A preliminary study illustrating some limitations as useful references in protein expression studies

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So can I just don't use a loading control
Maybe show the commasi blue stained gel as a reference to show the loading of each lane are the same.
I just trust the protein concentration I got.

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If you don't want to do quantitative western blot coomassie or ponceau s might be enough.
Have you been searching for publications dealing with same research interest and looked weather they use a loading control or not?

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I plan to use ErK2 as loading control in WB .

The following information is from
The Protein Kinase Facts Book by Grahame Hardie and Steven Hanks
Academic Press, 1995

Erk1/2 is ubiquitously distribued in tissues and cell lines with largest amounts in the nervous system. It is developmently regulated. Erk1 and Erk2 differe in subcellular distribution although both are found in the cytoplasm, in nuclei and are associated with membranes and the cytoskeleton. These are activated by phosphorylation of tryosine and threonine residues. The higher enzymatic activity occurs when both sites are phosphorylated.

Can I deduce that:
The proteins are consitutitively expressed. They are activated (phosphorylated) by MEK. The total amount of protein is not increased with activation, only the amount of phosphorylated protein.

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If you are facing the choice of beta-actin and total actin, which one is less possiblly changed?

Why so many people using beta-actin?

In my research, I clearly see the actin system was regulated by the drug I use.

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