Rabbit His-Tag Antibody

January 28, 2019

BlueskyReddit
Department of Biology
Memorial University
Ph.D. Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Thermo Fisher Scientific

Product Name:

6x-His Tag polyclonal Antibody

Catalog Number:

PA1-983B

Our main goal is to purify an N-terminus-histidine tagged protein and perform a binding assay to check its binding with a ubiquitous secondary molecule cyclic-d-GMP. For that, I performed a pull-down assay using streptavidin beads and tested the eluted product by running SDS-PAGE. Since my protein is His-tagged, I used this 6X-His Tag antibody for detecting the protein of interest. The main reason for particularly using this His tag antibody was the secondary antibody we use in our lab is from rabbit and will be compatible with this product.

Experimental Design and Results Summary

Applications

Western Blot

Sample

E. coli

Primary Incubation

1:250 ( overnight 4°C, slow on shaker)

Blocking Agent

5% skim milk

Secondary Incubation

1:1000

Tertiary Incubation

N/A

Detection

ECL substrate

Results Summary

The band intensity is very high with 1:250 dilution ( suggested dilution by company), so I increased the dilution to 1:500 and it worked well. I even reused the same antibody solution for 2 more membranes within a week time and worked well. Note: Showed multiple non-specific binding as well.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

It is a very good polyclonal antibody and worked well for the first attempt.

The Bad

Showed multiple non-specific bindings as well

The Bottom Line

Works perfect for the detection of His-tagged proteins with high specificity but also gave few non-specific bands as well.

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