Inconsistent Clarity

November 28, 2018

BlueskyReddit
Oncology
Johns Hopkins School of Medicine
Lab Technician

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signaling Technology

Product Name:

HIF-1α (D2U3T)

Catalog Number:

14179

We used this antibody for western blot on T cells to see the levels of HIF-1-alpha in different glutamine concentration conditions. We were interested in whether the glutamine conditions could affect the cells' expression of HIF-1-alpha in a hypoxic condition. The antibody worked sometimes but was not always consistent.

Experimental Design and Results Summary

Applications

Western Blot

Sample

T cells

Primary Incubation

1:2000 overnight in 4°C

Blocking Agent

5% nonfat dry milk

Secondary Incubation

Anti-rabbit IgG HRP-linked

Tertiary Incubation

N/A

Detection

ECL Invitrogen

Results Summary

The detection was inconsistent across the times we used the antibody. It was not very strong specificity and it would detect nonspecific bands outside of the 120 kDa range. Sometimes it did give us clear bands; however, it was never consistent.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Easy to use and sometimes clear bands

The Bad

There were some nonspecific bands

The Bottom Line

The results could be clear, but there are times where we weren't sure what bands we were looking at. CST suggested 1:1000 dilution in BSA; however, we found that doing 1:2000 or 1:2500 was best for T cells. Overall, it was useful, but there might just be some nonspecific bands.

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