PURIFICATION OF UBIQUITIN AND PRODUCTION OF SPECIFIC ANTIBODY DIRECTED AGAINST IT

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HOJAT MAHSHID; SHABANI ASHRAF; TALEBIAN A.; AKHOUNDI MOHAMMAD MAHDI; GHODS R.; SADEGHI M.R.

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Journal: IRANIAN JOURNAL OF BIOLOGY Year:2006 | Volume:19 | Issue:3 Page(s): 251-263

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Title

PURIFICATION OF UBIQUITIN AND PRODUCTION OF SPECIFIC ANTIBODY DIRECTED AGAINST IT

Author(s)

Keywords

UBIQUITINQ3

PURIFICATIONQ2

ANTIUBIQUITIN ANTIBODYQ3

FLOURSCEIN ISOTHIOCYANATEQ2

Abstract

UBIQUITIN is a small protein of 76 amino acid residues that has attracted the concern of researchers of different science due to its multiple functions in the most biological process. It contributes in process such as cellular proteolysis, replication control and gene expression, maintenance of chromatin structure, cell differentiation, gametogenesis and etc., which shows numerous function of this vital protein. The immunochemical methods (ELISA, radioimmunoassay, Immunofluorecence) are sensitive and rapid techniques for the analysis of UBIQUITIN and other such biological molecules which all use specific antibodies as detector. Therefore preparation of specific antibody directed against UBIQUITIN is a valuable tool in detecting and assessing of this protein. In this study we attempted to purify UBIQUITIN from human red blood cells and produce specific antiUBIQUITIN antibody and conjugate these antibodies in order to employ in immunochemical studies. In this study UBIQUITIN was first purified from packed blood cells by several steps including rapid denaturation of protein at 90°c, precipitation of UBIQUITIN and other protein in 90% saturated ammonium sulfate and chromatography on DEAE-Sephadex. In the next step to increase the immunogenecity of UBIQUITIN, cross linking of this protein to form polyUBIQUITIN was attempted by employing glutaraldehyde .The prepared immunogenic polyUBIQUITIN, were injected multiportally in to the rabbit .The reactive antibody was detected against polyUBIQUITIN, using enzyme linked immunoassay . In order to isolate rabbit serum antibody, Protein A affinity chromatography was employed .Free UBIQUITIN was coupled to CNBr-activated Sepharose 4B so that by passing rabbit antiserum over the column antiUBIQUITIN antibody was purified. Specificity of antibodies was also examined using enzyme linked immunoassay. Finally antiUBIQUITIN antibodies were conjugated with FLOURSCEIN ISOTHIOCYANATE. Results on SDS-Gel Electrophoresis of purified UBIQUITIN and antiUBIQUITIN antibody from Ion exchange column and affinity column appeared as a single band, indicating protein purity. It has also confirmed the specificity of purified antibodies referred to antiUBIQUITIN antibodies by using ELISA. Use of such PURIFICATION methods gave a yield of 1mg UBIQUITIN from 100ml packed blood cell and 0/6 mg antiUBIQUITIN antibodies from 8 ml immune serum. Study and identification of UBIQUITIN function, play an important roles in biological research therefore preparation of antiUBIQUITIN antibodies as an initial tool of these researches in immunochemistry studies is very important and there has been much interest in the production of these antibodies. The offered method in the preset study for PURIFICATION of UBIQUITIN and antiUBIQUITIN antibodies is a reliable way to achieve this aim. Access to Antibodies conjugated with Fluorochrome molecules in this study; provide a tool in UBIQUITIN studies by Immunofluorecence methods.

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APA: Copy

HOJAT, MAHSHID, SHABANI, ASHRAF, TALEBIAN, A., AKHOUNDI, MOHAMMAD MAHDI, GHODS, R., & SADEGHI, M.R.. (2006). PURIFICATION OF UBIQUITIN AND PRODUCTION OF SPECIFIC ANTIBODY DIRECTED AGAINST IT. IRANIAN JOURNAL OF BIOLOGY, 19(3), 251-263. SID. https://sid.ir/paper/21377/en

Vancouver: Copy

HOJAT MAHSHID, SHABANI ASHRAF, TALEBIAN A., AKHOUNDI MOHAMMAD MAHDI, GHODS R., SADEGHI M.R.. PURIFICATION OF UBIQUITIN AND PRODUCTION OF SPECIFIC ANTIBODY DIRECTED AGAINST IT. IRANIAN JOURNAL OF BIOLOGY[Internet]. 2006;19(3):251-263. Available from: https://sid.ir/paper/21377/en

IEEE: Copy

MAHSHID HOJAT, ASHRAF SHABANI, A. TALEBIAN, MOHAMMAD MAHDI AKHOUNDI, R. GHODS, and M.R. SADEGHI, “PURIFICATION OF UBIQUITIN AND PRODUCTION OF SPECIFIC ANTIBODY DIRECTED AGAINST IT,” IRANIAN JOURNAL OF BIOLOGY, vol. 19, no. 3, pp. 251–263, 2006, [Online]. Available: https://sid.ir/paper/21377/en

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