I am looking for phospho-KIT (Y719) and total-KIT on mast cells using western blot.
I can detect beautiful bands with phospho-KIT but it is impossible to detect total-KIT whatever I do!
- After membrane stripping using two antibody (Cell signaling & santa Cruz) => no band
- On a new membrane using two antibody (Cell signaling & santa Cruz) => no band
- On a new membrane using a new antibody (Cell signaling) => no band
All the steps of WB are working good, so where is the problem?
It could just be that your anti-phospho KIT antibody is much better than the anti-total KIT antibodies that you have.
A second possibility is that the signal from the total Kit antibody is weaker and you may have to go to as high as a 1:100 dilution to detect signal.
Hi Siham, I do not know whether I understand right. When I want to detect them (total protein and phosphorylated protein) on the same membrane, I will use the weak antibodies first and have a longer exposure time for it; after that I would strip the antibodies from the membrane, block the membrane again and use the second antibodies. If you want to have beautiful results, you could adjust the amount of secondary antibodies. Thanks.
Best,
Shuaipeng
Hello Siham,
We stain KIT and pKIT on a daily basis in our lab, however in GIST cell lines. For total KIT we use the one from DAKO 1:1000 - gives beautiful bands. For pKIt we use either p703 or 719, both from CS, 1:1000.
I wouldn't stain both on the same membrane (as they are the same species), but rather use "sister" membranes, same lysates on two gels. Of course you could strip, check stripping success by secondary only and then re-stain. However with these strong stains we sometimes have problems getting rid of them completely by stripping...
Hope that helps
...and just a general consideration: I, myself only use (i.e. buy) SC antibodies if I really really have to, i.e. if they're the only ones offering it. Then you have to be lucky (sometimes very lucky) to pick a working clone....
Dear all,
Thank you for your answers.
Usually, I use the same antibodies, pKIT (Y719) and KIT from CS. I always stain first with pKIT then I do membrane stripping to stain with total KIT. It always works very well, I had beautiful bands with both pKIT and total KIT.
However, these last weeks, I had a problem with total KIT, I do not see any bands.
I thought that I should not do membrane stripping, so I stain a fresh membrane with a fresh batch of KIT antibody (CS), and I had no bands. So I tried an other antibody from santa cruz, I had the same result. At the end, I thought that the antibodies were too old, so I ordered a new antibody from CS (as usually). I tested it on a fresh membrane but I had no bands.
All my reagents work well with pKIT and other proteins.
Thank you for your help !
As suggested in earlier responses, looks like the problem is with your total KIT antibody, while the phospho one is working perfect. Try another antibody or get back to the company for replacement. Sometimes, they have problems with a specific lot number
We had also problems with the SCBT antibodies before. Changing the lot (we tested some) finally helped.
the best ways
A) Immunoblotting of whole cell extract using an antibody that recognize specifically your phos-protein, if available
B) Immunoprecipitation with the antibody anti-total protein and immunoblotting against the anti-phos-tyrosine or anti-phos-Serine etc,
Since your antibody worked before, but gives no longer sufficient results, I would also recommend to order a new one. Maybe the old one was comprimized somehow. Be careful with SC antibodies, these are very LOT dependent! If you still want to try the old ones, enhance the concentration, and - as recommended by the others - start with the weak antibody and maybe even use an enhanced ECL.
To check your total antibody reactivity, run a WB with different concentrations of whole extract before to order a new one, e.g. 1 ug until 50 ug of protein. If your antibody not recognize none of these conditions, it is better order a new one (preferably cell signaling or abcam, I never had a good experience with SC).
Good luck!
Hi Siham Bibi,
1. Did anyone of your lab members have similar problem? If so maybe you should also consider whether the membrane is usable. I also had this problem before but later it was the bad membrane that caused the problem. How about the second antibody? Does your second antibody out of date?
2. Did you treat the cells with some drugs which can strongly promote KIT phosphorylation? Sometimes, the drug can induce completely phosphorylation of the protein.
3. If not, maybe it is your primary antibody problem. But I think this is unlikely because you have used several antibodies from different company. Maybe you can ask others to do the immuno blot for you to see whether they have similar problem.
- Badges
- Science method