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High resolution one-dimensional electrophoretic separation and partial characterisation of human head hair proteins.
Electrophoresis. 1995 Jan; 16(1):110-5.E

Abstract

A reproducible, rapid procedure for the extraction, labelling and separation of human hair proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Hair proteins were extracted in 8 M urea, containing 0.2 M mercaptoethanol, followed by sonication. Extracts were neutralised with Tris and incubated with either labelled (14C) or unlabelled iodoacetic acid to S-carboxymethylate cysteine groups. Proteins were separated on 12.5% SDS-polyacrylamide gels and gels stained with Coomassie Brilliant Blue and/or silver nitrate to reveal major protein bands. Gels were then treated with a fluorographic agent, dried and autoradiographed to reveal major sites of S-carboxymethylation. A given gel was scanned by laser densitometry after Coomassie and/or silver stain to quantitate the protein content of each major protein zone. An autoradiogram of the same gel was scanned to estimate the cysteine content of each major zone. In this way it was possible to partially characterise rapidly and reproducibly many different protein zones in different individual samples on one gel at the same time. By calculating the ratio of autoradiograph absorbance to Coomassie Blue absorbance, protein zones could be assigned to four different categories, viz: very high cysteine (VHC) proteins, high cysteine (HC) proteins, low cysteine (LC) proteins and very low cysteine (VLC) proteins. The method described is reproducible, rapid and inexpensive enough to be suitable for mass screening. Overall the results were more informative than previously reported one-dimensional separations and indeed this technique may well be more suited to forensic and/or population investigations than the much more laborious and time-consuming two-dimensional techniques.

Authors+Show Affiliations

Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

7737084

Citation

Khawar, S L., et al. "High Resolution One-dimensional Electrophoretic Separation and Partial Characterisation of Human Head Hair Proteins." Electrophoresis, vol. 16, no. 1, 1995, pp. 110-5.
Khawar SL, Watson K, Jones GL. High resolution one-dimensional electrophoretic separation and partial characterisation of human head hair proteins. Electrophoresis. 1995;16(1):110-5.
Khawar, S. L., Watson, K., & Jones, G. L. (1995). High resolution one-dimensional electrophoretic separation and partial characterisation of human head hair proteins. Electrophoresis, 16(1), 110-5.
Khawar SL, Watson K, Jones GL. High Resolution One-dimensional Electrophoretic Separation and Partial Characterisation of Human Head Hair Proteins. Electrophoresis. 1995;16(1):110-5. PubMed PMID: 7737084.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - High resolution one-dimensional electrophoretic separation and partial characterisation of human head hair proteins. AU - Khawar,S L, AU - Watson,K, AU - Jones,G L, PY - 1995/1/1/pubmed PY - 1995/1/1/medline PY - 1995/1/1/entrez SP - 110 EP - 5 JF - Electrophoresis JO - Electrophoresis VL - 16 IS - 1 N2 - A reproducible, rapid procedure for the extraction, labelling and separation of human hair proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Hair proteins were extracted in 8 M urea, containing 0.2 M mercaptoethanol, followed by sonication. Extracts were neutralised with Tris and incubated with either labelled (14C) or unlabelled iodoacetic acid to S-carboxymethylate cysteine groups. Proteins were separated on 12.5% SDS-polyacrylamide gels and gels stained with Coomassie Brilliant Blue and/or silver nitrate to reveal major protein bands. Gels were then treated with a fluorographic agent, dried and autoradiographed to reveal major sites of S-carboxymethylation. A given gel was scanned by laser densitometry after Coomassie and/or silver stain to quantitate the protein content of each major protein zone. An autoradiogram of the same gel was scanned to estimate the cysteine content of each major zone. In this way it was possible to partially characterise rapidly and reproducibly many different protein zones in different individual samples on one gel at the same time. By calculating the ratio of autoradiograph absorbance to Coomassie Blue absorbance, protein zones could be assigned to four different categories, viz: very high cysteine (VHC) proteins, high cysteine (HC) proteins, low cysteine (LC) proteins and very low cysteine (VLC) proteins. The method described is reproducible, rapid and inexpensive enough to be suitable for mass screening. Overall the results were more informative than previously reported one-dimensional separations and indeed this technique may well be more suited to forensic and/or population investigations than the much more laborious and time-consuming two-dimensional techniques. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/7737084/High_resolution_one_dimensional_electrophoretic_separation_and_partial_characterisation_of_human_head_hair_proteins_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0173-0835&date=1995&volume=16&issue=1&spage=110 DB - PRIME DP - Unbound Medicine ER -