I want to raise primary antibody against my protein of interest in my lab. I need help to make it. I have full length mRNA sequence of my gene. Can you provide me any protocol to raise primary antibody?
Thanks in advance.
U have to clone the gene in a vector and then express and purify the reecombinant protein. This protein can be used as antigen. It has to be injected in mice or preferably rabbit using established protocols (primary dose, booster dose). After specific number of days kill animal and get antibody.
If you have gene you can go for recombinant DNA technology and get a expression of your protein interest . The same protein with adjuvants can be used to raise the antibodies in animal, as per your availability. You can schedule the process of immunization and antibody collection (serum). By half ammonium sulfate saturation you will get immunoglobulins. Afterwards you can collect your antibodies by affinity chromatography by tagging you protein in chromatography beads.
At what dose should I inject the recombinant protein for antibody generation???
Thank you all for your kind responses.
Usually you need between 50-100 ug/ml of antigen per animal.
Here you are a complete guidelines that can help you better understand the process
https://acuc.berkeley.edu/guidelines/antibodies.pdf
regards
We use to raise antibodies for our ES antigen in goat by immunizing intramuscularly with 500 µg protein/ mL with 1 ml Freund’s incomplete adjuvant on days 0, 20, 33 and 45. Immune sera were collected on days 32, 44, 57, 60 and thereafter fortnightly .
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