7 replies to this topic

[dcch]

Two months ago, I tried to immunise my rabbit with my protein of interest, hoping to produce rabbit polyclonal antibody for my study. When I obtained second boost serum and used it for western blot (1:200 in TBS) against 80ng of my purified protein of interest, I observed no band!!! I am afraid there was no antibody being produced...

The detail of my immunization: I run preparative SDS-PAG with 250ug of purified protein. Then I stained the gel with copper reverse stain. The band was then cut and minced with pastel and mortar (with about 2mL of PBS). The gel was injected subcutaneously into 6 location of rabbit back. Immunization was performed at 3 weeks interval. Two rabbits were being used for immunization, but both showed no result in my WB.

Is there any problem with my method? Has anyone in forum success in producing polyclonal antibody with my protocol above? If there is antibody being produced, should it be seen in 2nd boost serum?

It is very sad for not able to get antibody after taking care the rabbit for two months...it is even worst because I am unlikely to repeat it again (unless being approved by animal ethic committee again, which takes long long time...)

Any comments are welcomed...Thanks.

Edited by dcch, 01 January 2011 - 04:10 AM.

[K.B.]

I can't tell you for sure if that's the problem but the protocols I know has two steps your is lacking:
1. Fixing protein in gel using glutaraldehyde (2%, 60 minutes).
2. Freund adjuvant - complete for first dose, incomplete for second and subsequent.

I use ELISA to control immunization and compare pre- and post-immunization sera.

[Chelo]

dcch, on 01 January 2011 - 04:05 AM, said:

Two months ago, I tried to immunise my rabbit with my protein of interest, hoping to produce rabbit polyclonal antibody for my study. When I obtained second boost serum and used it for western blot (1:200 in TBS) against 80ng of my purified protein of interest, I observed no band!!! I am afraid there was no antibody being produced...

The detail of my immunization: I run preparative SDS-PAG with 250ug of purified protein. Then I stained the gel with copper reverse stain. The band was then cut and minced with pastel and mortar (with about 2mL of PBS). The gel was injected subcutaneously into 6 location of rabbit back. Immunization was performed at 3 weeks interval. Two rabbits were being used for immunization, but both showed no result in my WB.

Is there any problem with my method? Has anyone in forum success in producing polyclonal antibody with my protocol above? If there is antibody being produced, should it be seen in 2nd boost serum?

It is very sad for not able to get antibody after taking care the rabbit for two months...it is even worst because I am unlikely to repeat it again (unless being approved by animal ethic committee again, which takes long long time...)

Any comments are welcomed...Thanks.

Hi,
In our lab, we produce rabbit polyclonal Abs routinely. We immunize the animals 4 times, the first one using complete Freud adyuvant, the rest are performed with uncomplete Freud adyuvant.
I hope this helps.

[dcch]

So even with gel slice, it has also to be injected together with CFA/IFA?  

[Chelo]

dcch, on 01 January 2011 - 04:34 PM, said:

So even with gel slice, it has also to be injected together with CFA/IFA?  

Yes, although the best is to electro-ellute the protein from the gel (to get rid of the toxic acrylamide) and then to mix it with the adjuvant. Keep in mind the adjuvant helps to sensitize macrophages and other APC cells needed for the immune response.

[mdfenko]

Chelo, on 03 January 2011 - 03:29 AM, said:

Yes, although the best is to electro-elute the protein from the gel (to get rid of the toxic acrylamide) and then to mix it with the adjuvant. Keep in mind the adjuvant helps to sensitize macrophages and other APC cells needed for the immune response.

polymerized acrylamide (polyacrylamide) is not toxic. it is perfectly fine (although difficult) to inject polyacrylamide into the animal.

monomeric acrylamide is toxic.

[Chelo]

mdfenko, on 03 January 2011 - 08:07 AM, said:

Chelo, on 03 January 2011 - 03:29 AM, said:

Yes, although the best is to electro-elute the protein from the gel (to get rid of the toxic acrylamide) and then to mix it with the adjuvant. Keep in mind the adjuvant helps to sensitize macrophages and other APC cells needed for the immune response.

polymerized acrylamide (polyacrylamide) is not toxic. it is perfectly fine (although difficult) to inject polyacrylamide into the animal.

monomeric acrylamide is toxic.

Polyacrylamide is considered non-toxic. However, there are always acrylamide monomers trapped inside the acryl-bis mesh. In any case, this is not the point of the discussion.

[Prep!]

hey don get offended chelo... sometimes topic divertions are common and informative!!!