Okay Western Blot ALKBH5 Antibody with Some Background Bands

December 19, 2018

LMP
University of Toronto
Graduate Student

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Quality of Results

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Company:

Proteintech Group Inc

Product Name:

Anti-ALKBH5

Catalog Number:

16837-1-AP

Recent studies have demonstrated the impact of RNA modifications in cellular expression of genes. Here, I asses the expression of ALKBH5, which is an important mediator of the RNA modification N-6 methyladenoisne (m6A). ALKBH5 has been demonstrated to serve as an ‘eraser’ of m6A levels on RNA.

Experimental Design and Results Summary

Applications

Western Blot

Sample

A375 whole-cell lysate

Primary Incubation

1:2000, Overnight at 4°C

Blocking Agent

5% skimmed milk - 40 min room temperature

Secondary Incubation

1:10000, anti-rabbit HRP-conjugated antibody, 30 min at room temperature

Tertiary Incubation

N/A

Detection

ECL reagent - film

Results Summary

Antibody detects ALKBH5, but there are non-specific or isoforms of ALKBH5 that appear near the correct size of ALKBH5. This can make it difficult to determine the expression of ALKBH5. Approx. size 40–50 kDa. Strong background bands slightly above 50 kDa.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Can detect ALKBH5, but gels needs to be resolved properly.

The Bad

Observe a few non-specific bands near the correct size of ALKBH5 – makes it difficult to cleanly detect ALKBH5.

The Bottom Line

Probably look for other ALKBH5 antibodies that have lower background/non-specific bands.

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