Dear Alberto,

I have tried application of antibodies via pipette solution in the past. However I did not get any effect. I think the concentration or the dilution of antibody to be used, as in immunocytochemistry, depends on the quality of antibody. Usually with good antibodise you need to use very small amounts at 1:10 000 or 1:20 000 dilution. The antibody you have used may be working well and you may need to it dilute to at least 1:2 000. You mention you have observed some fast and strong effects. Are you waiting for passive diffusion of antibody or applying voltage pulses? You will need to do some controls by pre-incubating your antibody with the target peptide prior to application, and see if you can block the effects you observe.

Kind regards,

Refik Kanjhan

Hi Refik,

thank you very much for you answer. I am studying persistent firing so, from the membrane rupture I am running some protocols in voltage clamp to follow the effect of the antibody on persistent activity. And usually around 10mins I see an effect. 

I used the antibody at that dilution mainly because in that paper it worked for them at 1:100, even if as you pointed out, it's very very concentrated. 

In the meanwhile I used heat inactivated antibodies (30mins at 90°C) and the cell's activity was not affected (but I have to increase the number of cells to be 100% sure). Do you think it is usually better to inactivate the antibodies with the antigen or with heat?

In the next weeks I will try an higher antibody's dilution and see what happen.

Best,

Alberto

Hi Alberto,

we and others have used anti-Kv3 on channel antibodies successfully in our pipettes in the past. We wrote a review on such uses of antibodies that you might find useful:

https://www.ncbi.nlm.nih.gov/pubmed/16045997 .

Immunopharmacology--antibodies for specific modulation of proteins involved in neuronal function.

Dallas M, Deuchars SA, Deuchars J., J Neurosci Methods. 2005 Aug 15;146(2):133-48.

We have also made an on-line table of antibodies people have used like this (it may not be very up-to-date):

http://www.fbs.leeds.ac.uk/research/antibody/index.php 

Hope you find these useful.

Jim

Hi Jim,

thank you very much for your review, there are lots of useful info! Thanks also for the second link you posted.

I am getting now a clearer idea of the antibody's concentration that usually people use.

Best,

Alberto

Hi Alberto,

I think ideally you shoudl try both: inactivate the antibodies with the antigen and also with heat?

best wishes, Refik