2 replies to this topic

[EinsteinKid]

Hi All,

I am trying to validate 3 antibodies.

B65701R
B65420R
G5V119-500

Every protocol I read is saying I need secondary antibodies. Is there a way to validate it without secondary abs.
Can anyone please give me a link to such protocol. I am not a protein person. I work with DNA and RNA. So please help me on this matter.
Thanks in advance,

[Shilpa Dasari]

EinsteinKid, on Jul 24 2009, 04:26 PM, said:

Hi All,

I am trying to validate 3 antibodies.

B65701R
B65420R
G5V119-500

Every protocol I read is saying I need secondary antibodies. Is there a way to validate it without secondary abs.
Can anyone please give me a link to such protocol. I am not a protein person. I work with DNA and RNA. So please help me on this matter.
Thanks in advance,

Hello,

Yes, you need secondary antibody which will bind your primary antibody (unless your primary is already labelled for one step detection-in your case I think they are not). Selection of your secondary antibody depends on primary antibody. For example, first two antibodies were raised in Rabbit hence you can try Goat anti rabbit and for last one  Donkey anti Goat.
You can try a simple "Dot Blot" (google it for protocol) to validate your antibody and also to optimise your primay concentrations.
If you are going to do western blot, I have attached a protocol and beginers guide for you.


This is my first reply on this forum. Hope I have given a reasonable answer.
Hope this helps you and good luck

shilpa

Attached Files

•  WESTERN_BLOT_ABCAM.pdf   210.56K   95 downloads

Edited by Shilpa Dasari, 24 July 2009 - 12:03 PM.

[EinsteinKid]

Hi Shilpa,

Thank you so much for the reply. I will go for secondary antibodies.  I thought there is a way to do it without going for a secondary Abs.
I am going to search a protocol for dot blot.
Thanks again,