[原创]BCA 蛋白定量试剂盒

macgene 发表于 2009-1-5 09:34:00

Bicinchoninic Acid (BCA) Protein Assay KitBCA 蛋白定量试剂盒Kit Components:Reagent A <span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">（500mL<span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">）Reagent B <span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">（10mL<span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">）Protein Standard<span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">（BSA<span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">）：1mL <span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">（10mg/mL<span style="FONT-SIZE: 9pt; COLOR: black; LINE-HEIGHT: 150%; FONT-FAMILY: 宋体; mso-bidi-font-family: Arial-ItalicMT; mso-ascii-font-family: Verdana; mso-hansi-font-family: Verdana; mso-bidi-font-style: italic;">） Brief Description:
BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a one step procedure. A molybdenum/tungsten blue product is produced as with the Lowry. In addition to standard liquid handling supplies a visible light spectrophotometer is needed with transmission set to 562 nm. Glass or polystyrene (cheap) cuvettes may be used. The BCA is used for the same reasons the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace the Lowry because it requires a single step, and the color reagent is stable under alkaline conditions. Experimental Procedures:<p class="MsoNormal" align="left" style="MARGIN: 0cm 0cm 0pt 21pt; TEXT-INDENT: -21pt; LINE-HEIGHT: 150%; TEXT-ALIGN: left; mso-pagination: widow-orphan; mso-layout-grid-align: none; mso-list: l0 level1 lfo1; tab-stops: list 21.0pt;">Ø         Preparation of Standard working solution (SWR): Mix 100 volumes reagent A with 2 volumes reagent B. Note：The stock solutions are stable. The working solution is stable for 1 week and should be green. <p class="MsoNormal" align="left" style="MARGIN: 0cm 0cm 0pt 21pt; TEXT-INDENT: -21pt; LINE-HEIGHT: 150%; TEXT-ALIGN: left; mso-pagination: widow-orphan; mso-layout-grid-align: none; mso-list: l0 level1 lfo1; tab-stops: list 21.0pt;">Ø         Prepare protein standards containing 0.2, 0.5, 1, 2, 5, 10mg/mL BSA. Prepare a standard curve of absorbance versus micrograms protein (vice versa). <p class="MsoNormal" align="left" style="MARGIN: 0cm 0cm 0pt 21pt; TEXT-INDENT: -21pt; LINE-HEIGHT: 150%; TEXT-ALIGN: left; mso-pagination: widow-orphan; mso-layout-grid-align: none; mso-list: l0 level1 lfo1; tab-stops: list 21.0pt;">Ø         Prepare samples containing 0.2 to 10 mg/mL. <p class="MsoNormal" align="left" style="MARGIN: 0cm 0cm 0pt 21pt; TEXT-INDENT: -21pt; LINE-HEIGHT: 150%; TEXT-ALIGN: left; mso-pagination: widow-orphan; mso-layout-grid-align: none; mso-list: l0 level1 lfo1; tab-stops: list 21.0pt;">Ø         Add 1 mL SWR to each 20 microliters sample and mix. Incubate 30 min. at 37ºC. <p class="MsoNormal" align="left" style="MARGIN: 0cm 0cm 0pt 21pt; TEXT-INDENT: -21pt; LINE-HEIGHT: 150%; TEXT-ALIGN: left; mso-pagination: widow-orphan; mso-layout-grid-align: none; mso-list: l0 level1 lfo1; tab-stops: list 21.0pt;">Ø         Read at 562 nm. Color will be stable for at least one hour.<p class="MsoNormal" align="left" style="MARGIN: 0cm 0cm 0pt 21pt; TEXT-INDENT: -21pt; LINE-HEIGHT: 150%; TEXT-ALIGN: left; mso-pagination: widow-orphan; mso-layout-grid-align: none; mso-list: l0 level1 lfo1; tab-stops: list 21.0pt;">Ø         Determine concentrations of original samples from the standard curve. Comments:A longer incubation increases the sensitivity of the assay. The heating can be stopped earlier to prevent the color from becoming too dark. The assay can be performed at room temperature, but there is greater variability among proteins and the assay is less sensitive. Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

macgene 发表于 2009-1-5 09:34:00

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macgene 发表于 2009-3-30 14:43:18

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详情请登录网站：www.macgene.com，联系电话：010-82057786

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[原创]BCA 蛋白定量试剂盒