Using AlexaFluor 647 Labeling Kit To Conjugate A Monoclonal Antibody

June 19, 2017

Bloodworks NW
Research
Postdoctoral research fellow

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Company:

ThermoFisher Scientific

Product Name:

Alexa Fluor® 647 Protein Labeling Kit

Catalog Number:

A20173

Our lab generated our own monoclonal antibody and we used this kit to conjugate the antibody to Alexa 647 for flow cytometry analysis.

Experimental Design and Results Summary

Application

Flow cytometry staining

Starting Material

Purified monoclonal antibody

Protocol Overview

The protocol for this labeling kit can essentially be broken to 2 steps - incubating the antibody with the dye and purifying the labeled antibody from unincorporated dye.

Tips

For step 2.4. The mixture will enter the column resin but will not move along the column unless you add the elution buffer. Once the mixture are inside the column, add elution buffer right away.

Results Summary

We labeled 2 antibodies that day and one worked better than the other. Even though we used 1mg @2mg/ml as mentioned in the protocol, I suspect that concentration of one of the Ab was incorrect, resulting in poor labeling.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

It works!

The Bad

Very very slow flow through the column, even with elution buffer only at Step 2.4. So slow that we tried to repack the column many times but with not much improvement. Much easier if you just accept that the flow is going to be very slow..and be patient wait it out.

The Bottom Line

Yes, this kit works but be prepared to read through the convoluted instructions. It will be much easier second time around.

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