hi, all
I‘v been performing immunofluorescence, anti nestin(7A3) and nestin(2Q178), for a long time, and never have obtained a positive staining, these antibodies were bought from santa.
Is there any knack or key-step to get good results?
Xiangchun Ju
I can tell you just this much about SantaCruz antibodies:
I have bought about 15 from this company in the last 6 years. Only 3 work as they are supposed to. Two of them where against phosphorylated forms of proteins. The other one is for IHC. All of them are for western blot. The ones for westernblots estarted to give a signal only after I used a non-reduced sample.
Summary: Buy SantaCruz ONLY when someone that you know (publications don't count!) has used it successfully.
If you want an antibody for IHC / Flow cytometry , that will really work (99% of the cases), I would recommend BD Biosciences, Upstate or Millipore.
An extra tipp: From the antibodies that worked, one is the PY99. This one works ONLY when our solutions have LESS that 0,1% Triton. I think your Triton concetration is to high for the blocking buffer. Since is a seccion, I am not sure how good you can get rid of the excess triton in your sample before you add your antibody. Detergents are not that helpful in teh antibody reaction.
Good luck!
thanks,
I tried two concentrations of dilutedc nestin Ab, 1:50 & 1:200 in PBS with 1.5% normal goat serum (NGS) and 0.1% triton X-100, respectively.
My procedure is:
- Adult mouse was perfused with 0.9% saline and 4% PFA, than brain was removed and postfixed in 4% PFA for 2 hrs, at last brain was cryoprotected in 20% sucrose for 24hrs
- cryosectioning in 30um
- blocking buffer (10% NGS + 0.5% triton X-100) for 1hr or 30min at RT
- iprimary antibody for 2hr at 37C or overnight at 4C
- PBS for 5min X3
- Secondary antibody for 2hr at RT
- PBS for 5min X3
- mount and check
I've tried 4 blocking buffer according to papers:
1) 0.1% PBST, 5% NGS, 0.05% Tween-20, 1% BSA
2) 10% NGS, 0.5% Triton X-100
3) 0.2% Triton X-100, 3% BSA
4) 10% NGS, 1% Trition X-100
all didn't work.
is a dilution in 1:200 still very high?
is there any problem with these treatments?
or others?
I can tell you just this much about SantaCruz antibodies:
I have bought about 15 from this company in the last 6 years. Only 3 work as they are supposed to. Two of them where against phosphorylated forms of proteins. The other one is for IHC. All of them are for western blot. The ones for westernblots estarted to give a signal only after I used a non-reduced sample.
Summary: Buy SantaCruz ONLY when someone that you know (publications don't count!) has used it successfully.
If you want an antibody for IHC / Flow cytometry , that will really work (99% of the cases), I would recommend BD Biosciences, Upstate or Millipore.
An extra tipp: From the antibodies that worked, one is the PY99. This one works ONLY when our solutions have LESS that 0,1% Triton. I think your Triton concetration is to high for the blocking buffer. Since is a seccion, I am not sure how good you can get rid of the excess triton in your sample before you add your antibody. Detergents are not that helpful in teh antibody reaction.
Good luck!
thanks everyone!
having a conclusion, concentration of antibody and triton is the key factor. thanks!
Hi!
I am using also Santa Cruz´s antibodies. What I have really learnt from them is the following: it is very important that the results are shown in the datasheet. Avoid always the Abs without examples published: In the 99% of the cases, they do not work. Moreover, make sure that they have been used for your method (IHC, IF, WBs) etc....often they work just in IHC but not WBs or viceversa.
One think which really works with them is....complaining!!! If your Ab does not work you can easily obtain another in a short time :) (from another stock or better another kind with the same characteristics-I mean new sc.....!)
For human samples, I find excellent the Abs from ATLAS Antibodies (Sigma). They are polyclonal monospecific and until now they have worked fine in my hands.
In the other cases, Abcam or Millipore are quite good.
Good luck!
thank stella!
Thanks for your advice. I used antibodies from Millipore and Dako, and they work well. I've tried kinds of conditions including perfusion, fixation, cryosectioning, blocking, incubating-time & temperiture, secondary antibody...
Their service was quite good, and so I hoped to find the point to work with Abs of sc if I have enough time, just like Jody Gascon said "Some times it may take a little longer for them to develop ", yes!
thanks all the time.
Maybe not helping, but I thought it was interesting... It's a review of the employees of SC.. It may explain somehow few things...
http://www.glassdoor.com/Reviews/Santa-Cruz-Biotechnology-Reviews-E39955.htm
Good luck with your IFs!
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