I have used Akt as control (equal amount of protein loading) for p Akt . The blot showed no change in Akt level on all four lanes but p Akt level got increased in treated lane and decreased in untreated lane.
Does Akt mean Total Akt? i.e, Akt = Akt+pAkt
Akt = phosphorylated Akt + unphosphorylated Akt
The first problem is that the phosphorylation residues exist in several number. The antibody to detect p-Akt cannot detect all those residues.
The second problem is that persistent phosphorylated Akt-mediated mTOR signaling activation leads to the down-regulation of total Akt expression. However, as far as I read your question, that does not occur.
Hi,
your results seem suggesting that. treatment increases p-Akt levels, when compared to untreated (I believe it is your control).
Total Akt = unphosphorylated Akt + phosphorylated Akt
Regards,
Daniele
I have seen band shifts in total protein if there is any phosphorylation present but the intensities were more or less same. The band shift is due to addition of phosphate groups.
Yoshida, you are right about mTOR. But it is not true for all cell lines. Some cell lines show that downregulation and some don't.
quite often when treating cells with insulin i would see a decrease in total Akt levels and huge increases in p-Akt, more with longer periods of treatment ie hrs
Just depends on your cells and your treatment
From your answers i could understand and believe that pAkt antibody bind only to phosphorylated form of Akt. However Akt antibody can able to bind with both unphosphorylated and phosphorylated forms of Akt .Is this is one of the reason for unchanged level of Akt?
It depends on your treatment. In case where your drug increases only phosphorylation of the protein(AKT) then you will see an increase in phosphorylation but no change in total AKT. To confirm this, you can do qPCR and show no change in mRNA levels for AKT. One more thing is type of antibody you use. Certain company sells specific isoform AKT ab i.e AKT1,2,or 3. However most companies sells phosphorylated ab which is for all three isoforms. Hope this helps along with other answers.
You are doing the right experiment in that when using phospho-specific antibodies it is critical to use the cognate total protein antibody to show that the level of the target (phosphorylated protein) is unchanged (and is essentially a loading control). There are different phosphorylation sites on Akt/PKB including Ser473 and Thr308 (for Akt1/PKBalpha) and these sites are increased upon PI3K activation. Typically, protein kinase levels do not change in the short term. They are regulated by post translational modifications (usually phosphorylation) or by second messengers and changes are transient and reversible. Sometimes, phosphorylation causes a shift on SDS-PAGE that can be observed in blots of the total protein. Lastly, it is worth noting that phospho-specific antibodies can be misleading since the degree of phosphorylation is difficult to assess and these antibodies have quite different avidities/sensitivities. Just because there is a signal in a phospho-specific antibody blot does not mean the protein is highly phosphorylated (could only be a few percent).
We have seen changes in total Akt during Th17 cells differentiation in the presence of total Akt phosphorylation
I've been trying to find a good loading control for our cell model systems. We look at HIF1a deletion, CKB lof/gof, and drug treatment of CKB in normoxia and hypoxic environments.
I was validating a cell line and saw a HUGE decrease in total AKT in a HIF1a KO line under hypoxic conditions (not at normoxia).
Can anyone point me twd a signalling pathway that might explain this?
It's a Pan-AKT monoclonal. I could see a faint Phospho band, which may or may not be present in other samples(total band blows out in comparison)
Use beta Actin or tubulin as housekeeping protein. They should not decrease as total AKT did.
Beta Actin is affected by both hypoxia and drugs that bind actin/tubulin. These are issues for our models.
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