Rapid protein spot blotting assay

Summary

In routine protein spot blotting, the blotting membrane is closed for 1 hour, reacted with primary antibody for 1 hour, and then reacted with secondary antibody for 1 hour. To allow for lower sensitivity, the steps of closure, primary antibody, and secondary antibody can be combined to speed up the process, taking less than 15 minutes. This experiment was derived from the Protein Purification and Characterization Lab Guide by Houzhu Zhu.

Operation method

Rapid protein spot blotting assay

Materials and Instruments

Tris buffered salt solution (TBS) TBS+1% bovine serum albumin TBS+0.1% Tween-20(TBST) 5-bromo-4-chloro-3-indole-phosphate Azurotetrazolium Blue Tetrazolium (BCIP NBT) Color Developing Reagent Mouse Anti-σ32 Monoclonal Antibody 3RH2 (MAb 3RH2) Rabbit Anti-Mouse Immunoglobulin G (IgG)~Alkaline Phosphatase Splice
Nitrocellulose membrane

Move

Materials and equipment

Nitrocellulose membrane (Schleicher & Schuell, Inc.)

Mouse ^anti-σ32 monoclonal antibody 3RH2 (MAb 3RH2)

Rabbit anti-mouse immunoglobulin G (IgG) ~ alkaline phosphatase affix

Reagents

Tris buffered salt solution (TBS)

TBS+1% bovine serum albumin

TBS+0.1%Tween-20(TBST)

5-bromo-4-chloro-3-indole-phosphate/nitrogen blue tetrazolium (BCIP/NBT) color development reagent

(For the formula, see "Preparation of reagents", pp.184~189)

Operating procedure

1) Take a small square piece of nitrocellulose membrane and mark it with a grid and number. Place it on a piece of paper towel and wet the nitrocellulose membrane and paper towel with TBS.

2) Flatten the nitrocellulose membrane against the paper towel with a spatula or pipette tip, and dot a 1-ul sample on the nitrocellulose membrane at the position indicated by the grid.

3) Let the sample stand for 2 min after spotting, let the sample pass through the nitrocellulose membrane by capillary action, and then place the membrane in antibody/BSA solution (mouse anti-σ32MAb 3RH2 and rabbit anti-mouse IgG-alkaline phosphatase conjugate, each diluted 1/1000 in TBS+1% BSA solution).
Note: We have found that when samples contain excess polyethylenimine (PEI) (e.g., samples obtained by FEI precipitation at very high PEI concentrations), false-positive reactions can be induced and are mostly bound to MAb. Such false positives can be minimized by enclosing the blot in TBS+1% BSA for 2 min before incubation with the mixed Hangers.

4) Bind to the antibody for at least 6min under slow shaking, then aspirate the supernatant (save the sample for further blotting analysis), and wash the nitrocellulose membrane with TBST for two times quickly.

5) Wash the nitrocellulose membrane with TBST for 3 times, at least 1min each time, then rinse with water quickly and add BCIP/NBT color developer.

6)Spot color development 5~20mm,then rinse the nitrocellulose membrane with water,gently pat dry,and make it completely dry before including the experimental records.

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