Quantitative protein spot blotting assay

Summary

Protein blotting, which uses immunological methods to measure the amount of a particular protein. This experiment was derived from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.

Operation method

Quantitative protein spot blotting assay

Materials and Instruments

Mouse anti-σ32 monoclonal antibody Horseradish peroxidase covalently labeled sheep anti-mouse immunoglobulin G ECLTM#1 and #2 Reagents Sodium dodecyl sarcosinate Tris-buffered salt solution
Nitrocellulose membrane Spot blotting device ECLTMHYPERFILM or Kodak X-ray negative buffer A

Move

Materials and equipment

Samples, obtained from each purification step

Nitrocellulose membrane (0.45um, 8x11.5 cm) (Schleicher & Schuell, Inc+)

Spot blotting device (MinifolundefinedI, Schleicher&Schuell, Inc. 27510)

Mouse ^anti-σ32 monoclonal antibody 3RH2 (MAb3RH2)

Horseradish peroxidase covalently labeled sheep anti-mouse immunoglobulin G (IgG-HRP) (AmershamLifeScience, Inc.)

ECLTM#1 and #2 reagents (AmershamLifeScience, Inc.)

ECLTMHYPERFILM (AmershamLifeScience, Inc,) or Kodak X-ray Negative

Reagents

Buffer A

Sodium dodecyl sarcosinate (SKL) (20% storage solution)

Tris-buffered salt solution (TBS)

TBS+0.1% Tween-20 (TBST; prevents binding of non-specific proteins)

TES+1% bovine serum albumin (BSA)

(For formulation, see "Reagent Preparation", PP.184~189)

Operating Procedures

1) Prepare a multiple dilution series of the most important reagents from each purification step according to the following scheme. Take 20ul of each sample and add 180ul of Buffer A+0.3% SKL for dilution. After thorough mixing, take 100ul of each dilution and add it to 100ul of Buffer A (one pipette tip for each dilution), and then make 8 successive doubling dilutions. Multiple dilutions can be conveniently performed on a microtitre plate, which was incubated with 100 TBS+1% BSA for 10 min and rinsed with TBST before use. Eight wells per sample were spiked with dilutions of 20, 40, 80, 160, 320, 640, 1280 and 2560 times. The component samples to be analyzed were: Sample A (crude lysate), Sample B (0.2% DOC supernatant), Sample C (first 2% DOC wash), Sample D (inclusion bodies solubilized in SKL), Sample E (FEI supernatant), Sample F (0.3mol/LaCl wash), Sample G (0.9mol/LaCl eluate), Sample I (immunoaffinity chromatography (IAC)), and Sample B (0.9mol/LaCl eluate). immunoaffinity chromatography (IAC) on-column sample], Tube 3 fraction of the IAC column, Sample J (dialyzed SKL extract), peak fraction of the POROS 50Q column, and a ^σ32 standard of known concentration (~0.5 mg/ml).
Note: The first dilution with Buffer A + 0.3% SKL is to dissolve the inclusion bodies in Sample A. The first dilution with Buffer A + 0.3% SKL is to dissolve the inclusion bodies. Failure to dissolve the inclusion bodies can result in isolation of ^σ32 from the solution, which in our experience underestimates the amount of ^σ32 in Sample A by a factor of eight!

2) Pre-wet the nitrocellulose membrane in Milli-Q water for 2 min, then in TBS for 5 min. Attach the blotting device, making sure that the plywood is mounted tightly so that the sample does not leak from one spiked well to the other (first a rectangular piece of filter paper, then a rectangular piece of nitrocellulose membrane).

3) Add 40ul of each dilution (20x to 2560x) to the appropriate well (for one dilution series, if you start with the thinnest, you may not change the pipette tip). Allow to stand for 3 min, then slowly evacuate. Wash 3 times with 100ul of TBST (stop vacuuming and start again when adding liquid).

4) Remove the nitrocellulose membrane from the blotting device. Incubate in TBS+1%BSA for 30 min, then in 14 ml-anti solution (14 ml TBS+1%BSA and 5ulMAb 3RH2) for 1 h. Pour off the MAb solution, store at 4°C and reuse within a few days.

5) Wash the nitrocellulose membrane with TBST 4 times for 5 min each time.

6) Incubate the nitrocellulose membrane in 14 ml of secondary antibody solution (14 ml TBS+1% BSA and 7ul goat anti-mouse IgG-HRP) for 1 h. Decant the secondary antibody solution and store for reuse. Wash the nitrocellulose membrane with TBST 4 times for 5 min each time.

7) Mix 7 ml ECL#1 reagent and 7 ml ECL#2 reagent (i.e., 0.15 ml mixture/cm2 nitrocellulose membrane and pour onto the membrane. Mix well, incubate for 1 min, and discard excess reagent. Wrap the cellulose nitrate membrane in Saran wrap. Expose the ECL Hyperfilm for 2, 10, and 60 s. Develop the negative (2 min in developer, 0.5 min in rinse water, 2 min in fixer, 10 min in rinse water). The negatives were dried.

8) The negative is quantified by scanning or visually estimated to compare which dilution of each sample shows a signal comparable to that of the standard. For example, if the ^σ32 Standard shows a medium density signal at 640x dilution and Sample D shows the same density at 1280x dilution, the concentration of Sample D can be estimated to be twice that of the ^σ32 Standard (see Figure 3-4).

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