hello all,
I have done western on A549 cell protein lysate. .I found the total AKT in the treated group was less compared to the control group.
How to conclude from this result?
many thanks
I suppose you have made sure you loaded the same absolute amount of protein for each well/lane? That's critical for drawing conclusions in assays like these.
You could even include a 2nd reference, like B-actin, GAPDH or a-tubulin.
If you did match input correctly then it's well possible that your treatment might affect the total amount Akt present in your cells. Perhaps not the effect you were interested to see, but it could be an effect of your treatment and should be interpreted as such if you'd ask me.
What steps to take from here is up to you or your PI...
It's most honest to present the data as is, but many will express Akt-phosphorylation as pAkt/tAkt or when checking multiple compounds normalize all conditions to 1 control treatment. Usually to obfuscate inconsistencies in total Akt/Erk/etc.
Did you load equal amount of proteins on each well. only the phospho protein levels generally varies( upon equal loading of sample). In certain cancers i have read the total protein also changes inspite of equal protein loading . You could also try using beta actin as loading control and validate your results
are you sure you load the same amount of protein for all lanes?in generally total akt level is not changing. How about your loading control?
thank you all for your responses... ys i make sure of loading equal amount of protein an i do have b-actin too to quantify this.
Dinesh Kumar Parandhaman , yes i too came across some papers, just talking about total protein.
If this result is rigid, replicable and found after normalization to an adequate standard, reduced Akt expression may reflect reduced Akt-dependend signaling (since less protein may lead reduced phosphorylation signaling (in sum), further leading t o reduced Akt-dependend cellular effect in your lung cancer cells (e.g. reduced pro-survival signaling, reduced expression of down-stream transcriptional events etc.).
However, other explanations are surely possible, too.
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