Lots of Non-Specific Bands

Genetics
Yale
Tech

Overall

Quality of Results

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Company:

Abcam

Product Name:

Anti-CDYL antibody

Catalog Number:

ab5188

Our lab generates a lot of protein knockouts in mouse embryonic stem cells using crispr-cas9 technology. We use western blots and subsequent sequencing to screen potential knockout clones. We chose this antibody because it had good reviews with rat cell lysates and was chip grade.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Whole cell lysates from mouse ES cells

Primary Incubation

1:500 dilution overnight in 5% milk buffer at 4C

Blocking Agent

5% milk in PBST

Secondary Incubation

1:3000 anti-rabbit secondary at RT for 1 hour

Tertiary Incubation

NA

Detection

Exposure with SuperSignal™ West Femto Maximum Sensitivity Substrate, imaged with apparatus from Bio-Rad

Results Summary

Results show a lot of bands--hard to tell which is specific. We confirmed one of these clones (#4) as a true knockout through sequencing, and the WB kind of confirms this since the staining is lighter than the other lanes. However, there is still the issue of multiple bands.

Additional Notes

None

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Summary

The Good

Works

The Bad

Lots of nonspecific bands

The Bottom Line

It works, but isn't very specific. Other methods would be needed to confirm presence/absence of the protein.

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