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In-gel total protein quantification using a ninhydrin-based method.

Amino acids (2013-07-10)
Sung Ung Kang, Seok Heo, Gert Lubec
RESUMEN

Precise in-gel quantification of total protein amount of bands or spots in gels is the basis of subsequent biochemical, molecular biological and immunological analyses. Though several methods have been designed to evaluate relative amounts of proteins, these methods are of limited reliability because (semi-) quantifications depend on the amount of protein migrating into the gel and different proteins may lead to different absorptions/intensities of stained bands or spots. In the present study, we described a method to quantify both, hydrophilic and hydrophobic proteins using in-gel digestion with proteinase K, subsequent extraction and acid hydrolysis followed by the use of the ninhydrin reaction. The protocol is accurate and compatible with mass spectrometric characterization of proteins. Reproducible in-gel protein quantification was performed from SDS-PAGE and IEF/SDS-PAGE gels using bovine serum albumin as a standard protein. Bacteriorhodopsin separated on SDS-PAGE gel was quantified in addition in order to show that the method is also suitable for quantification of hydrophobic protein. This protocol for reliable in-gel protein quantification, which not only provides "arbitrary units of optical density", can also be completed in a minimum of 4 days or maximum 1 week depending on the type of electrophoresis with the disadvantage of being time consuming.

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Sigma-Aldrich
Proteinasa K from Tritirachium album, buffered aqueous glycerol solution, for molecular biology, ≥800 units/mL
Sigma-Aldrich
Proteinasa K from Tritirachium album, lyophilized powder, ≥30 units/mg protein
Sigma-Aldrich
Proteinasa K from Tritirachium album, Reagents designed and manufactured under current ISO 13485 certification.
Sigma-Aldrich
Proteinasa K from Tritirachium album, ≥3.0 unit/mg solid, lyophilized powder
Sigma-Aldrich
Proteinasa K from Tritirachium album, ≥500 units/mL, buffered aqueous glycerol solution
Sigma-Aldrich
Proteinasa K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology