I have only been performing normal defection of total proteins and is wondering what changes I should make for detecting phosphoproteins?
1) do I wash cells with 1x tbs instead of pbs before lysing.
2) do I use TBS instead of PBS in lysis buffer recipe?
3) should I block in 3% BSA and dilute Primary antibody and secondary antibody in 1%bsa?
4) for detecting phosphorylated form of protein do I incubate with PRImary antibody overnight, detect next day, strip, block in MILK then incubate with antibody against total protein for 2 hours?
5) is it safe to dilute antibody against total proteins with milk after detecting phosphoproteins?
1). You should wash the cells with PBS only.
2). Both are suitable for the cells but if you aren't getting the results with PBS that time you can use TBS.
3). 3% BSA is sufficient for blocking or 5% Non-fat milk powder, you can use.
4). Yes, you should incubate primary antibody overnight and 2 hrs is sufficient for the secondary antibody.
hi sameer thanks for your reply.
for qn 4, i was asking if i should incubate with primary antibody (for total proteins) overnight or is 2hrs enough? i am not sure if overnight is too long since i already incubated with primary antibody (against phosphoprotein) overnight the first time around. Will the membrane antigens degrade or something?
also i was advised on some forums to not use PBS for detecting phosphoproteins since the antibody can react against the phosphates. Please give me your take on this.
many thanks!
Hi Iwan,
Using TBS is also recommended for phosphoproteins . Overnight incubation is necessary if the antibody is phospho-SER, THR or TYR but if it is a phosphoprotein-specific antibody, then it can work even in 2 hrs incubation.
Stripping and re-probing with total antibody, you can do with either milk or BSA but in my opinion, stick to one blocking agent for convenience sake.
Regards,
Atul
If you are looking for phosphorylated proteins, use TBS-T since the phosphate in PBS might interfere with the binding of your antibody.
For the rest: just try... it always depends on your antibody.
I agree with the Teresa Otto, and probably the PBS may be interfering, even in the lysis buffer.
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