Labeling the Probe for Chemiluminescent EMSA

November 13, 2015

BlueskyReddit
Texas Tech University Health Science Center
Internal Medicine
Postdoctoral Researcher

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Company:

Thermo Scientific

Product Name:

Biotin 3' End Labeling Kit

Catalog Number:

89818

This kit is used to label the probe which is used for chemiluminescent EMSA. Basically, it uses TdT to incorporate the 1-3 biotinylated ribonucleotides at the 3' ends of the DNA strands which is further used in the binding reaction for performing EMSA.

Experimental Design and Results Summary

Application

Chemiluminescent EMSA method

Starting Material

Mouse/Human cell lysate

Protocol Overview

This kit is used to label the 3' end of the probe. The 3' end of the probe is labelled with biotin and it is less likely to interfere with the hybridization or sequence-specific binding of proteins. It requires 5pmol of 3'OH ends in each reaction. In this procedure the biotin-labeled double-stranded DNA and the complementary oligos should be end-labeled separately and then annealed them before use by using PCR and should be kept it room temperature until the binding reaction is ready for EMSA.

Tips

Label the oligos seperately and anneal them before use.

Results Summary

This Biotin 3' end labeling method is non istotopic. The enzyme terminal deoxynucleotidyl transferase (TdT) is responsible for catalyzing the deoxynucleotide incorporation onto the 3'-OH end of the DNA0. It is generally used for labeling the single stranded DNA but it will also label the duplex DNA with 3' overhangs and blunt duplexes.

Additional Notes

None

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Summary

The Good

It works great and the results are reliable.

The Bad

It is a time consuming process.

The Bottom Line

This kit makes it very easy to label the probe. It can be used in a variety of applications like EMSA, Northern blots, Southern blots, in situ hybridizations, and colony hybridizations.

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