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The GST-fusion protein eluted from the GSH-sepharose is diluted and need to be concentrated. Combine eluted fractions (El1+El2) in 50 ml falcon tube, freeze at -80oC, close with puncture plastic cap. Lyophilize over-night. Resuspend in 0.5-1 ml of DDW, spin, and transfer to a small tube. Store at -20oC.
Rabbit Screening
It is important to screen pre-immune sera from several (2-6) rabbits on western blots containing plant protein extract (soluble, and membrane pellet). Often pre-immune sera cross (at 1:500 dilution) reacts strongly with plant proteins.
Preparation of protein for antibody production
Depending on the antiginicity of your fusion protein, a range of 20-100 mg of fusion protein is required for first injection.
NOTE: When using GST-fused to small peptide (10-20 aa) use ~250-500 mg of protein/injection.
Boost: For second injection (a month later 5-20 mg of fusion protein is required/boost injection).
Two rabbits are being used to generate antibody to the same antigen. One rabbit is injected with the native form of GST-fusion protein, and the second rabbit is injected with SDS-reduced denatured form of GST-fusion protein.
1. For the native form the GST-fusion protein sample (normally 100 mg in 0.5 to 1 ml) is mixed with equal volume adjuvant (see below).
2. For the denatured form the GST-fusion protein sample (normally 100 mg in 0.5 to 1 ml) is mixed to 1% SDS, 5 mM DTT, and incubated for 15 min at 65oC, cooled and then mixed with adjutant (see below). Alternatively, the fusion protein can be separated on reducing SDS-PAGE, the protein is eluted from the gel, mixed with adjuvant and injected to rabbits.
At the day of injection
To 0.5 ml of protein sample (native or denature formed) add 1 ml of Titer Max, vortex. Add additional 0.5 ml of protein solution. With a all plastic (3 ml) syringe equipped with 20 gauge needle pipette up and down the content until it become very viscous.
Injection into rabbit
Up to 2 ml of TiterMax:protein sample is injected /rabbit.
Treatment of pre-immune and immune sera
1. Before injection to the selected rabbit (it is preferred to inject samples into 2 rabbits) collect blood for pre-immune.
2. 28 days from initial injection, collect first bleed (~30 ml), cool at 4oC for several h.
At the same day of collecting blood inject a boost of your protein
3. Centrifuge at 2,000 g for 15 min at 4oC. Transfer the clear supernatant into new tube.
4. Add Na-azide from a 20% stock to final 0.02%. Divide the clear blood (5-15 ml) into 1 ml aliquots, and freeze -20oC. Keep one tube at 4oC (up to several months) for analysis.
5. 28 days from second injection, inject another sample, and collect at the same day the second bleed (~30 ml). Cool, spin, aliquot as in steps 3, 4.
6. If the titer is low, a third, and fourth injection can be done. Blood is collected and sera is analyzed see below.
7. Collect the entire blood from the rabbit (~150-200 ml = 4th and final bleed).
Analyze each pre-immune sera and various bleeds from 1st, 2 nd, 3rd, 4th at 1:500 to 1:2000 dilution in TBST-5% milk on western blots containing 50 mg each of total, soluble and membrane fraction from plants (Arabidopsis, Tobacco, Maize, Rice, etc.); and 1 mg of GST, and GST-fusion proteins produced in E. coli.
Antibody can be further purified if needed (See Bar-Peled and Raikhel (1996), Anal. Biochem.)