Endy:Victor3 plate reader/Calibrating the GFP-separated label: Difference between revisions
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==Materials & Methods== | ==Materials & Methods== | ||
===Purified GFP=== | ===Purified GFP=== | ||
As a gold standard, we obtained a purified sample of GFP from [[Jennifer Braff]] and the [[Sauer Lab]] (all of which should be folded due to the purification process). The concentration of GFP in the sample had been measured using a [[nanodrop]] and was ~120μM. [[Jennifer Braff]] had diluted the GFP into pH 5 Hsub>2</sub>O water. Since we needed the GFP to be correctly folded we needed to dilute the concentrated stock into a neutral pH buffer. I believe we used TE('''check''') | As a gold standard, we obtained a purified sample of GFP from [[Jennifer Braff]] and the [[Sauer Lab]] (all of which should be folded due to the purification process). The concentration of GFP in the sample had been measured using a [[nanodrop]] and was ~120μM. [[Jennifer Braff]] had diluted the GFP into pH 5 Hsub>2</sub>O water. Since we needed the GFP to be correctly folded we needed to dilute the concentrated stock into a neutral pH buffer. I believe we used TE('''check'''). | ||
===Experimental Samples=== | ===Experimental Samples=== | ||
Revision as of 10:02, 4 June 2006
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Introduction
It would be desirable to be able to relate the relative fluorescent measurements that we obtain from the plate reader to actual molecule numbers per cell. This would facilitate comparison of results from the plate reader with measurements from other machines. You can read more about standardizing GFP measurements here. This page describes the process to calibrate GFP counts using the GFP-separated label on the Endy lab plate reader to GFP concentration.
Approach
We chose to calibrate the GFP counts from experimental samples to a concentration of purified GFP in the well of a 96-well plate. It is not possible to do this calibration directly, as the quantum efficiency of purified GFP in solution may be different from GFP in vivo. The approach we took was to first calibrate GFP from experimental samples to the GFP counts of lysed samples from the same original culture. Next, we measured a standard curve relating the concentration of purified GFP in non-fluorescent cell lysate to GFP counts. These two sets of calibration data can then be combined to produce a calibration from GFP counts for experimental samples to GFP concentration.
Materials & Methods
Purified GFP
As a gold standard, we obtained a purified sample of GFP from Jennifer Braff and the Sauer Lab (all of which should be folded due to the purification process). The concentration of GFP in the sample had been measured using a nanodrop and was ~120μM. Jennifer Braff had diluted the GFP into pH 5 Hsub>2O water. Since we needed the GFP to be correctly folded we needed to dilute the concentrated stock into a neutral pH buffer. I believe we used TE(check).
Experimental Samples
Lysis Method
We looked into a few different ways of lysing the cells without denaturing too much of the GFP. We settled on B-Per II sold by Pierce as it seemed to be a quick and easy protocol that was sufficiently gentle to not denature the protein overly. Sean Moore had good experience with B-Per II and gave us a modified protocol based on the manufacturers protocol. We made some small modifications to that protocol to get reproducible results in our hands.
