Precipitation of Hevea brasiliensis latex proteins with trichloroacetic acid and phosphotungstic acid in preparation for the Lowry protein assay.
Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 microgram ml-1). In addition to its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 micrograms) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay. A modified Lowry microassay is described which enables about 3 micrograms ml-1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 microgram ml-1 protein present in 6 ml of solution can be assayed.
Yeang
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Biotechnology, Biochemistry, Molecular and Cell Biology Division, Rubber Research Institute of Malaysia, Kuala Lumpur.
Yusof
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F
Abdullah
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L
D011232:Chemical Precipitation; D003840:Deoxycholic Acid; D007840:Latex / Q000737:chemistry*; D010047:Ovalbumin / Q000032:analysis; D010772:Phosphotungstic Acid; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D012408:Rubber / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000032:analysis; D012972:Sodium Hydroxide; D012995:Solubility; D014238:Trichloroacetic Acid
D007840:Latex; D010940:Plant Proteins; D003840:Deoxycholic Acid; D010772:Phosphotungstic Acid; D012710:Serum Albumin, Bovine; D012972:Sodium Hydroxide; D014238:Trichloroacetic Acid; D012408:Rubber; D010047:Ovalbumin