NAD+/NADH concentration determination

March 13, 2015

University of South Florida
CMMB
Graduate Student

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Company:

BioVision

Product Name:

NAD+/NADH Quantification Kit

Catalog Number:

K337-100

One of our bacterial mutants shows a strong deregulation of core metabolic processes in RNA sequencing experiments. In order to confirm these results, we have employed a variety of biochemical as well as phenotypical analyses. Besides the determination of cellular stress proteins via Western Blot, we also analyzed the ATP levels of the cell and furthermore searched to assess NAD+/NAHD levels as marker. The results of the latter helped to validate the sequencing results, as well as better understand the deregulated processes within our mutant.

Experimental Design and Results Summary

Application

Determining intracellular NAD+/NADH concentration

Starting Material

Bacterial culture

Protocol Overview

1. Growth of bacteria overnight 2. Isolation of cellular fraction via Lysostaphin treatment and subsequent sonication 3. Spinning to remove cell debris 4. Following standard protocol supplied with the kit

Tips

Repeat in at least triplicate, better in larger groups. Especially for small variations, the typical error rate of the assay can prevent from getting significant results

Results Summary

As mentioned within the abstract, we were able to confirm our RNA sequencing results and are planning to prepare a manuscript in the near future

Additional Notes

None

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Summary

The Good

Very sensitive

The Bad

Variations from sample to sample, maybe due to high sensitivity of the product (see "The good"). Sample size could be smaller, to use less of the reagent

The Bottom Line

A good product, that helped us throughout our research and that I would recommend to a colleague

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