Protein A Sephorose CL-4B for Immunopreciptation Application

September 16, 2015

Texas Tech University Health Science Center
Internal Medicine
Postdoctoral Researcher

Overall

Quality of Results

Ease-of-Optimization

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Company:

GE Health Care

Product Name:

Protein A Sephorose CL-4B

Catalog Number:

17-0780-01

We generally used Agarose A beads to pull down a particular protein of interest by immunoprecipitaion.

Experimental Design and Results Summary

Application

Western blot/Immunoprecipitation

Starting Material

Mouse cell lysate

Protocol Overview

We generally use 500 micrograms of protein along with a 2 microgram concentration of the specific antibody and allow it to incubate in the shaker overnight then the next day we add the Sepharose A beads and allow it to incubate in while shaking for 2hrs and wash two times with RIPA buffer and 1 time with PBS. We then remove the supernatant and add 2x loading dye and heat in boiling water for 5 min and then load for polyacrylamide gel for western blot.

Tips

Washing the beads should be done properly otherwise background will be increased.

Results Summary

Our protein of interest's bands were detected. Proteins which we wern't able to detect by western blot or proteins which present in low abundance can be detected using this immunoprecipitation method.

Additional Notes

None

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Summary

The Good

It's very easy to optimize the technique and the results are very reliable.

The Bad

If you do not wash the beads several times, it will give dark background.

The Bottom Line

These Sepharose A beads are very good for immunoprecipitation, we have to use these beads based on our source of the IgG of the antibody we are going to use and if your protein of interest is the same size as your IgG, then it becomes hard to differentiate. In that case you have to use another source of antibody. This product is really helpful to detect the low abundance proteins.

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