Estimation Of Protein From Sds-page

[Praveena SP]

Hello,

Good Morning, eveybody. I want to know the concentration of protein separated on SDS-PAGE i.e. estimation of protein present in the sample. What are the methods that we can follow for the estimation of a specific protein in a sample.

If anybody knows, please give reply.

[kmunson779]

The best way I have found to estimate protein amount in a band on SDS-PAGE is to run a standard curve of a purified protein of known concentration, i.e. BSA. In part of the gel, run serial dilutions of BSA whose concentrations cover the likely range of your protein of interest. In the same gel, run one or two dilutions of your sample. Stain with Coomassie Blue and image the gel. By eye, you can compare your sample band to the BSA bands and get a range or estimation of concentration, or you can scan/take a picture of the gel and use a program like ImageJ to measure band intensity, plot the BSA standard curve, and find where the intensity of your unknown falls on that standard curve.

This will give you an estimate of concentration -- it's not quite as quantative as a Bradford or Lowry assay, but it does work better than a Bradford if your sample is not pure. The more points in your standard curve, and the more sample dilutions you use, the better the quantitation. The caveats for this method include that BSA may take up dye at a different ratio than your protein of interest, pipetting error plays a role as well as leakage from the wells, and your staining needs to be consistent across the gel.

[Praveena SP]

Thanku very much for ur reply. Can u please let me know whether that Imagef software is free or patented one.

[admin]

ImageJ is free.

[ashokvardhand]

Originally Posted by Praveena SP

Thanku very much for ur reply. Can u please let me know whether that Imagef software is free or patented one.

cleare information please give sir

[Foyjur]

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Accurate comparison of the intensities of different protein bands requires that the bands be within the linear dynamic range of detection, which is the range of concentrations from the faintest band that can be detected to the most intense band for which the signal is not saturated. When used to detect a Western blot containing a serial dilution of transferrin protein
Quantify chemiluminescent Western blots over a wide dynamic range
WesternBrightTM Quantum
WesternBright Quantum is a new chemiluminescent reagent specially formulated for CCD imaging. This novel Horseradish peroxidase (HRP) substrate provides a strong, long-lasting signal, the broadest useful linear range and high sensitivity for the most quantitative chemiluminescent Western assays.

The broadest linear range for the most powerful quantitationAccurate comparison of the intensities of different protein bands requires that the bands be within the linear dynamic range of detection, which is the range of concentrations from the faintest band that can be detected to the most intense band for which the signal is not saturated. When used to detect a Western blot containing a serial dilution of transferrin proteinQuantify chemiluminescent Western blots over a wide dynamic rangeWesternBrightTM QuantumWesternBright Quantum is a new chemiluminescent reagent specially formulated for CCD imaging. This novel Horseradish peroxidase (HRP) substrate provides a strong, long-lasting signal, the broadest useful linear range and high sensitivity for the most quantitative chemiluminescent Western assays.