Hi guys,
I am hoping to get some advice / insights into something I'm seeing in the lab. So I'm activating Jurkats with anti-CD3 and anti-CD28 antibodies for 0-10 minutes. After cell lysis I perform a BCA, acetone precipitation and run a Western Blot to probe for p44 and Lck in both their phosphorylated and 'total' states.
Now, for both of those proteins I see changes in phosphorylation depending on the length of activation as I would expect. But with the length of activation, also the total expression of those proteins seems to decrease - so total Lck and total p44 decrease when the cells are incubated for longer than 1 minute with the activating antibodies.
Now, the amount of protein loaded should be the same (BCA and acetone precipitation performed beforehand) and when I probed for gamma-tubulin to see whether it was an issue with the loading I saw good, even bands.
i haven't been able to find anything similar in the literature - normally the total protein used as loading control is very even (even for activation times much longer than mine) but I've consistently been seeing this effect since I started.
Is there any mistake or issue with the protocol that could make me see this effect? Or has someone ever seen something similar in Jurkats?
Hi Sigrun
I know that LCK is degradated after some time but the period is longer than the ones you are using. Is there a particular reason why you are precipitating your samples? The main problem is that you can never guarantee that your recovery was 100%, some protein may not precipitate and some may be lost in the washes. If your sample is too diluted you should try using less lysis buffer or a concentrator tube (and quatify afterwards). Sometimes the endogenous band (actin or tubulin) are so strong that we can't differentiate real intensity visualy. I believe you should not precipitate your sample and use a pixel quantification software to analyze your bands.
Hi Sigrun,
In my experience with LCK activation in human T cells, as well as some other phosphorylated ligands such as LAT, is that the antibodies probing TOTAL expression become gradually incapable of picking up LCK (or LAT) due to gradual increased phosphorylation status. Some antibodies/protein interactions are affected more than others. In essence, the antigen (LCK) starts to look different, and the affinity to the antibody decreases.
This effect becomes apparent in the immunoblot as "pseudo reduced total expression". For this reason we actually normalize our immunoblot data based on Actin or Gapdh levels and not Total protein expression. This observation is well known in our laboratory and other T cell signaling labs.
As Andre said, degradation would not happen within this time period. If I were you I would look at your loading with Actin, Gapdh, or some other loading control.
Hope this helps
Mahmood
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