7 replies to this topic

[Flint]

Hi, everyone.
Routinly, I perform IF staining by incubate the cryosections with diluted primary antibody at RT overnight. Now I want to reuse my antibody, so I want to know how long the diluted primary antibody have it's binding capacity. I dilute 1st Ab with PBS supplimented with 0.05% tween-20.

Thank you for your help!

Edited by Flint, 14 January 2011 - 08:07 AM.

[lab rat]

It will be fine two or three uses, I think. This is based on the experience of someone leaving the working tube on the counter overnight.

[sgt4boston]

Why not place it in the refrigerator when done?  The tween-20 is the critical factor as it will degrade the ab over time.  Also, if you wish to dilute your conjugates to 'working dilution' then you should consider using commercial stablizers/solutions or at least including some carrier protein such as BSA.

[Flint]

lab rat, on 14 January 2011 - 06:39 PM, said:

It will be fine two or three uses, I think. This is based on the experience of someone leaving the working tube on the counter overnight.

thank you so much for giving a hand. I will try and share the results.

[Flint]

sgt4boston, on 17 January 2011 - 03:39 AM, said:

Why not place it in the refrigerator when done?  The tween-20 is the critical factor as it will degrade the ab over time.  Also, if you wish to dilute your conjugates to 'working dilution' then you should consider using commercial stablizers/solutions or at least including some carrier protein such as BSA.

Many thanks, friend.
After use, I will put the diluted 1st Ab back into -20. and including BSA is a good idea. How about 0.1% BSA?

[sgt4boston]

0.1% BSA is fine.  Just remember you can not use abs that contain bsa for coating plates.  Only use bsa for bulking up conjugates or in other steps not involved with coating or coupling reactions.

[Flint]

Many thanks to valubel suggestions of Veteran. The primary Ab which have been used once (inbubate at RT overnight first time) have shown wonderfull staining result at second time. I believe they can still be used for at least 2-3 times. good news!

Edited by Flint, 22 January 2011 - 02:57 AM.

[beth]

Sodium azide helps to preserve diluted antibodies.

I personally never like to reuse antibodies when they have been already used in an immunoassay. The highest affinity Abs will have already been bound to the sections and therefore taken out of the mixture. Also, leaving the diluted Ab at room temperature for so long could result in degradation of the antibody and also evapouration of the liquid, therefore altering the Ab concentration. Personally, I think this makes results less reproducible. But I know a lot of people do this, so I guess it's personal choice...!