I am getting ready to analyze fish larval body protein by Bradford method. Recently I found NaOH is good to prepare protein solution by the sample. But I still have few questions.. 1.Is it necessary to add 100 ul water to dye regent when use the blank. If the dye regent is used without 100ul of H2O in blank, is it an issue? Because I already diluted my bio-rad dye regent four times with de-ionized water as per instructions. 2. At protein quantification by UV-1800, do we need use cubic calibration curve instead of linear calibration curve? 3. In the calibration curve, do we need to select the zero intersect or not? 4. Actually to prepare 2 ml of protein solution (100 ul will be pipette out as the sample to add with 5 ml dye regent later), how much mg of dry sample or wet sample do we need to take ?
I send you the protocol for larvae extract. Additionally, Its very importat to consider if you need to measure protein concentration in Brush border.
Yours
HN
1. Yes
2. The response is quadratic
3. Must have intersect
4. You need to dilute your sample until it enters your calibration curve
More importantly, Bradford's method is semi-quantitative and highly sensitive to systematic errors. . Do not expect to much from it.
Mr. Hector, It seems that during the extraction of protein, you advise to use mannitol with tris and secondly Cacl2 and then centrifugation at 9000g (RTF). finally, take the supernatant (100ul) for using with Bradford dye reagent. Is it?
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