I am trying to immunize a mouse with proteins but the proteins are proving hard to purify. I want use SDS-PAGE gel to seperate the proteins and immunize the protein with gel. How can I do this? The gel slice contains SDS, so that it does not affect the mouse. Can it make the antibody efficient?
I am struggling to understand your question? Can you please explain a bit more what you are trying to achieve?
Hi. Maybe you can try elute your protein from the piece of gel. This you can make it, washing the piece of gel with NH4HCO3 to remove the commassie from the gel and after that, you wash with aceto nitrile to dehydrate to remove you protein from the gel. And this help to remove the SDS. All this can be done in an eppendorf tube.
Maybe, if you immunize you protein together with the piece of gel, you could change the immunogenecity of your antigen, because the SDS and polyacrilamide work together like an adjuvant.
Next: this is a reference to remove protein from the piece of gel.
Picariello G, De Martino A, Mamone G, et al. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Mar 20;833(1):101-8.
I have immunized mice with proteins excised from SDS-gels. I excise unstained proteins (stain lanes with the same protein in a lane on both sites) carefully from the gel and try to have high protein concentration in the band. I crush the gel by injecting x 5 through a 1 ml syringe without a needle and then through the same syringe with a 21G needle. After crushing I mix the gel fragments with 0.2-0.5 ml PBS and use for direct immunization or to emulsify with an adjuvant.
http://www.ijabpt.com/pdf/9254-Chaturvedi[1].pdf
Depending on the properties of your protein it may be possible to remove the SDS from your solution maybe by reversing your phases in a column allowing the aliphatic tails of the SDS to interact with the polar mobile phase and be washed through then recover your protein.
The answers you have so far give some clues on what to do. First, do not stain the lane with the protein band that you want to use. Run 2 lanes side by side with the same protein-containing loads. Cut the gel between the lanes and stain one lane so that you can tell where the protein band is in the unstained piece of gel. Cut out the region of gel that should have the unstained protein. Chop up the gel into small pieces and put into a dialysis bag containing about 300 uL of Tris-Glycine PAGE running buffer. Seal the bag. To do this next step, you will either have to have or find someone who has Western Blotting Electrotransfer chamber/system. Suspend the dialysis bag (i.e. via a suture thread) within the cassette holder and place it into the chamber of the Western Blotting Electrotransfer system containing Tris-Glycine buffer. Run the system at 100 volts for up to an hour to elute the protein from the gel into the solution within the bag. Take ALL of the fluid from inside the bag, add whatever adjuvants, etc. you need for immunization and inject into the mouse. The samll amount of SDS that was on the protein will not affect the mouse or the immunization. In fact, it might only enhance the immunization effeciency.
I would just make one comment on some of the suggestions. It was unexpected to me that injection of the ground up polyacrylamide gel into the animal was proposed. Apart from the SDS, does anyone have an opinion on whether intravenous placement of greater than micrometer sized particles into a live, conscious animal for days-weeks is humane practice? The crosslinked acrylamide particles/fragments will not be broken down to soluble forms in the mammal and will certainly lodge in the capillaries of areas such as the brain, where such a load size would be relatively significant in a mouse.
Yeah, Leslie, I agree with you. That experiment I was carried out 8 years ago just one time before I entered the present institute. At that time, I followed my previous adviser's protocol to prepare antiserum for plant viral protein. We did muscle injection for rabbit but not intravenously for mice. I think your method is much better and make less pain for experimental animals.
Thanks Yixiang for the discussion. I think then we agree that isolating the protein from the gel prior to immunization is advised.
If you know the specific band on an SDS-PAGE gel, cut the bands and use ethanol method to precipitate/elude the proteins from gel. This protocol is available at www.kendricklabs.com/EtOHppt_2008.pdf. Once you've got you precipitated/eluded protein, you can as well lyophilized it and dissolve it appropriately with adjuvant, I prefer Sigma. You can adjust the volume with 1XTBS buffer. Do not exceed 1 ml of you don't have 1mg lyophylized sample. This sample is good for peritoneal injection closer to the hind-limb. Here, the fluid goes smoothly without stressing the animal or triggering excessive agitation. I hope its helps.
Here is a file how to do ethanol elution of proteins. I guess it helps.
Hi Jinji
Here you go this article
I hope it is helpful
Grettings
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