Question (70894) | Anti-Nestin antibody [SP103] (ab105389)

Go to datasheet (ab105389)

Question


Inquiry: I ordered the RabMAb Anti-nestin antibody and I tested it using conditions recommended in the spec sheet for ICC/IF (1:200). I used this antibody at the same time as some other nestin antibodies from other companies, so I know the cells (human glioblastoma stem cells) express a lot of nestin. And I was also testing the RabMab anti-GFAP antibody (ab68428) at the same time, and this antibody worked really well. I have since repeated the experiment using different antibody concentrations and still have negative results (I get no signal). I am using the Alexafluor goat anti- rabbit 488 secondary antibody and again I was testing other rabbit antibodies at the same time and it worked at the concentration I used (1:250). My protocol is: 1. Fix in 8% PFA added directly to cells in culture media in a 1:1 ratio so it's diluted to 4% 2. wash 3. Permeabilize with 0.1% Triton-x-100 4. wash 5. Block in 10% goat serum 6. Add primary antibody in blocking solution for 1 hr at RT (I've gone as low as 1:100) 7. wash 8. Add secondary antibody in blocking solution for 1 hr at RT 9. wash 10. Add Hoescht 33342 I tried to be as detailed as possible, but please let me know if you need anymore info about my experiment. Thanks for your help.

Answer

Thank you for contacting us. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab105389. I would also appreciate if you can confirm some further details:

1) How long did you permeabilize with Triton?

2) Overnight incubation was used in the image on our datasheet. This is usually the best way to get a strong signal in your cells.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Sign up