GST-fusion protein injection into rabbits

GST-FUSION INJECTION INTO RABBITS

Last Update: December 2006

PROCEDURE - Concentration of GST-fusion protein eluted from GSH affinity column

The GST-fusion protein eluted from the GSH-sepharose is diluted and need to be concentrated. Combine eluted fractions (El1+El2) in a 50 mL falcon tube, freeze at -80^oC, close with puncture plastic cap. Lyophilize over-night. Resuspend in 0.5-1 mL of dH₂O, spin, and transfer to a small tube. Store at -20^oC

PROCEDURE - Rabbit Screening

It is important to screen pre-immune sera from several (2-6) rabbits on western blots containing protein extracts (soluble, and membrane pellet). Often pre-immune sera cross reacts strongly with the protein source of interest (at a 1:500 dilution)

PROCEDURE - 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 the first injection

NOTE: When using GST-fused to small peptide (10-20 aa) use ~250-500 mg of protein/injection

Boost: For the second injection, 5-20 mg of fusion protein is required to boost the immune response. An injection will be required a month after the first injection

Two rabbits should be used to generate antibodies against 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, 5mM DTT, and incubated for 15 minutes at 65^oC, 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

PROCEDURE - Injection

To 0.5 mL of protein sample (native or denature formed) add 1 mL of Titer Max and vortex. Add an additional 0.5 mL of protein solution. Using a plastic (3 mL) syringe equipped with 20 gauge needle pipette up and down the content until it become very viscous

Up to 2 mL of TiterMax:protein sample can be injected into a rabbit

1. Before injection to the selected rabbit (it is preferred to inject samples into 2 rabbits) collect blood for pre-immune

2. 28 days after the initial injection, collect the first bleed (~30 mL), cool at 4^oC for several hrs

NOTE: during the same day that the first bleed is collected, inject a boost of your protein

3. Centrifuge at 2,000 xg for 15 min at 4^oC. Transfer the clear supernatant into a new tube

4. Add Na-azide from a 20% stock to a final concentration of 0.02%. Divide the clear blood (5-15 mL) into 1 mL aliquots and freeze (-20^oC). Keep one tube at 4^oC (up to several months) for analysis

5. 28 days after the second injection, inject another sample, and collect at the same day the second bleed (~30 mL). Cool, spin, and aliquot as in steps 3 and 4

6. If the titer is low, a third, and fourth injection can be done. Blood is collected and sera is analyzed

7. Collect the entire blood from the rabbit (~150-200 mL = 4th and final bleed or later if desired)

Analyze each pre-immune sera and various bleeds from 1st, 2nd, 3rd, and 4th bleeds 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 pertinent protein sources; and 1 mg of GST, and GST-fusion proteins produced in E. coli as positive controls

8. If the antibody is not clean enough for downstream applications, it can be further purified (see Affinity purification of antibodies (121))