Problem with pRb phosphospecific WB - Problem with pRb phosphospecific WB (Apr/18/2006 )

Dear friends, We are having problems with a phosphospececific antibody (WB). Your help and experience would be very very wellcome:

We are using this kit from Cell signalling: PhosphoPlus® Rb (Ser780, Ser795, Ser807/811) Antibody Kit . We have done several attempts all unsuccesfull. We are working with the protocol provide by the company. I guess we can improve something in order to get good results. I’m going to tell you exactly the protocol:

1. We prepare Whole cell extract (WCE) from mice embryos by lysis in NP-40 buffer (plus Complete mini (antiprotease) and NaF and Sodium Ortovanadate) and sonication (5seconds 30%). We have used this extract with other fosfoespecific antibodyes (like anti cdc2tyr 15 and cdc2 the161 and the results are good so I gues there is no problem with the extracts.
2. We load 60 ug of this extract and run in a 10% acrilamide gel.(mini protean III- Bio-rad)
3. Wet transfer (1h 100volts Nitrocellulose (Pro-Tran)). We check by Ponceau and by marker (prestained) and the transfer is good.
4. We block on TBST 5% milk for 1 hour.
5. We wash the membrane 3 times 5 minutes with TBST
6. We incubate O/N 4ºC with your antibodies Ser780, Ser795 or Ser807/811) diluted 1/500 in 5% BSA
7. Wash 3 times TBST
8. We incubate RT in TBST 5% BSA with your secondary antibody (anti-rabbit) 1/1000 for 1 hour.
9. Wash 3 times TBST
10. Incubate for a minute on Lumiglo (9ml MQ water 0,5ml Solution A 0,5 ml solutionB).
11. Eliminate the Lumiglo and we develop by normal AGFA films or Kodak BioMax films

As negative controls we use Serum starved Knockout cells for some Cdks (it is almost imposible to have Rb phosphorilated on this background) and also the WCE dephosphorilated (10 Units of CIP for 60ug of WCE incubated 1h 37ºC).

After all this we see bands but only non specific bands (they are also in the negative controls). We would like to get your advice in order to improve this results. We think that maybe it is better to block the membrane in BSA and maybe it is also better to run the gels in 8% acrilamide instead of 10%.


Thank you very much for all of you in advance,

hi
due to this great forum, i recently changed milk for BSA.
phospho specific westerns in my hands work when blocking and incubate with TBS T 2% BSA.
AVOID : PBS and milk (which as said by mfdenko contains lots of phosphoproteins).

you should see this thread

Thank you very much for your comment. I have also read that milk contains phosphoproteins wich relays in higher background, but the point is that Cell signalling recomends to block with 5% milk. In the past I have tryed both blocking agents (5% milk & 5%BSA) and I really didn't see any diference, but maybe I should try again. Do you have any experience with Rb phosphoespecific antibodies?

Thank you very much,
Antonio

only with Stat1 phosphorylated and other exotic phosphorylated proteins but no, not with pRb