I have produced IgG polyclonal antibodies in animal models by vaccinating them against an antigen. Now, I want to purify the whole IgG antibodies within the serum and then read the concentration of purified IgG for my further experiments. Does anybody know how can perform these two experiments?
P.S., As I do not have access, I prefer to carry out these two experiments without using my specific protein.
Hello Hasan,
to purify whole IgG I would recommend purification via a ProteinA- or ProteinG column (depending on the animal model you're using). The concentration of the obtained elution solution can be checked via photometry or HPLC while using a decent reference solution.
For purity check you can use SDS-PAGE, MS or HPLC.
If you want to get a picture of it's performance you will definitely need your antigen.
Isn't it weird that you don't have your vaccine antigen anymore?
Good Luck!
Marco
Thanks a lot for your detailed answer. Have you worked with ProteinG/A columns, suggesting me the Cat No. and its level of efficacy? I need to purify the desired IgG from just 10 ml rabbit serum.
https://www.sigmaaldrich.com/technical-documents/articles/biology/affinity-chromatography-antibodies/performing-purification-hitrap-protein-g-hp-columns.html
HiTrap® Protein G HP is quite reliable.
good luck!
Couldn't agree more with Ozkan Gelincik .
The GE columns have high capacity and are a good choice for purifying whole serum.
Good luck with your work!
Marco Wilke
Ozkan Gelincik
Thanks for your comments. Whats your idea about the kit "NAb™ Protein A Plus Spin Kit, 1 mL; Cat No. 89978".
https://www.thermofisher.com/order/catalog/product/89978
Sure. If you don't have the oportunity to use an FPLC then these Spin Traps are always convenient.
I usually use Protein A-sepharose to purify IgG from rabbit or human serum, and Protein G-sepharose for mouse antibodies. I have also used a mixture of Protein A/G-sepharose when uncertain about the antibody isotype. I use 1-2ml syringes to make columns for small scale preparations. Bound IgG is eluted with Glycine-HCL, pH 2.8, and neutralized immediately with bicarbonate buffer, pH 9.0. I have also used citrate buffer (pH 3.0) for eluting IgG from Protein A-sepharose columns or for affinity purification. After dialysis (PBS), the preparations are concentrated by centrifugation (50kDa cut-off). There is usually some loss of protein during the process. For protein A purified IgG fractions, I normally quantify the eluted protein by measuring absorbance at 280nm. For other total protein quantification, including cell lysates, I use BCA. The standard BSA and target proteins should be diluted in the same buffer and I use PBS. Chemicals such as DDT often used in various buffers effect protein quantification by assays like BCA.
Nasim Yousaf
Nice job!
Do you have any idea about the estimated level of total IgG (mg/ml) in well vaccinated (a prime + two boosts) as well as control one? I did search many, but found little.
Rabbit serum IgG concentration range is 4 – 10 mg/ml. I obtained about 4.5mg of IgG when 1ml of pre-immune rabbit serum was used for protein A-sepharose purification procedure. IgG was eluted with Glycine-HCL, pH 2.8, and neutralized immediately with Tris-HCL, pH 9.0 buffer.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies