Quantitative SDS gel staining and scanning

Summary

Although most proteins bind approximately the same amount of dye per milligram, there is still a 10% or greater variation in the ability to bind dye between proteins. Therefore, the values obtained by this method are not very accurate unless you can use the same protein as a standard to determine the protein concentration. In general, however, CBB staining is much more accurate than silver staining. This experiment is from the Laboratory Guide for Protein Purification and Identification, by Zhu Houzhu.

Operation method

Quantitative SDS gel staining and scanning

Materials and Instruments

CBB Staining Solution Decolorizing Solution
SDS-PAGE Electrophoresis device Gel scanning device

Move

installations

SDS-PAGE electrophoresis device

Gel Scanning Unit

Reagents

CBB Staining Solution

Decolorizing solution

(For the recipe, see "Preparation of Reagents", PP.184~189)

Procedure

1) Carefully add a set of protein samples in ascending order of amount to the SDS gel lane, and then add a set of pure protein samples in the same order as a standard for quantification. Perform electrophoresis as usual (see Appendix 5).

2) Remove the gel from the electrophoresis unit, rinse it in a decolorizing solution for 1 min to remove some of the SDS, and then soak it in CBB staining solution, shaking it slowly at 20°C (as on a decolorizing shaker). 0.75-mm-thick gels need to be soaked for lh, and 1.5-mm-thick gels need to be soaked for 4 h. The gel should be soaked in CBB staining solution for 1 min.

3) Pour out the CBB dye solution and soak the gel in the decolorizing solution, shaking slowly for 1~4 h, depending on the thickness of the gel (use a sponge or Kimwipe tissue to absorb the free dye). When most of the dye has diffused out of the gel, the absorbent is removed from the container and CBB dye is added to the decolorizing solution to achieve an _A650nm value of 0.1 (light blue). At that time, the concentration of CBB was greater than its average binding coefficient to the protein. The gel was equilibrated in this dilute staining solution overnight and stored in it until the scanning assay.

4) The stained and destained gel is scanned at 650nm and the amount of bound dye in each protein band is determined.

5) For each band, the amount of bound dye is plotted against the amount of sample, and the concentration of the target protein in each sample is calculated by comparing the slope of the resulting line with the standard curve.

6) Integrate the entire gel scan of the raw material or the purified product in each step and calculate the proportion of protein in the ^σ32 band to the total amount of protein.
Note: This value is the maximum estimate of purity. Often there are many bands below the limit of detection that cannot be ignored when added together. Most researchers often overestimate the purity of their protein preparations.

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