借问一个问题: 定量很低浓度的蛋白

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借问一个问题---定量很低浓度的蛋白
请教一个问题:

各位朋友:我用一篇文章中介绍的BCA微量法测定微量蛋白浓度,结果发现其漏了一项NaHCO3 于是加入相应量的NaHCO3, 结果线性关系还是不好, 而且不稳定, 所以想找原始文献, 但我一直找不到原始文献:

不知那位用过这种方法请指点指点,或者手头有这篇文献提供参考, 谢谢!

Schoel, B., Welzel, M., and Kaufmann, S. H. E. (1995) Quantification of protein in dilute and complex samples–modification of the bicinchoninic acid assay. J. Biochem. Biophys. Meth. 30, 199–206. From"the protein protocol handbook"

e-mail: zcd991@hotmail.com

附文章提供的试剂配方:

2.1. Standard Assay

1.Reagent A: sodium bicinchoninate (0.1 g), Na2CO3·H2O (2.0 g), sodium tartrate (dihydrate)(0.16 g), NaOH (0.4 g), NaHCO3 (0.95 g), made up to 100 mL. If necessary, adjust the pH to 11.25 with NaHCO3 or NaOH (see Note 1).

2. Reagent B: CuSO4·5H2O (0.4 g) in 10 mL of water (see Note 1).

3. Standard working reagent (SWR): Mix 100 vol of regent A with 2 vol of reagent B. The solution is apple green in color and is stable at room temperature for 1 wk.

2.2. Microassay

1. Reagent A: Na2CO3·H2O (0.8 g), NaOH (1.6 g), sodium tartrate (dihydrate) (1.6 g), made up to 100mL with water, and adjusted to pH 11.25 with 10 M NaOH.??????

2. Reagent B: BCA (4.0 g) in 100mL of water.

3. Reagent C: CuSO4·5H2O (0.4 g) in 10mL of water.

4. Standard working reagent (SWR): Mix 1 vol of reagent C with 25 vol of reagent B, then add 26 vol of reagent A.

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