關於實驗需要詢問的問題

第一點  ELISA盤子的底部到底是否會影響到吸光問題?實務上可以探討

BCA Protein Assay Reagent (bicinchoninic acid) 
Used in more labs than any other detergent-compatible protein assay.

 

BCA ... start with the basics

BCA…start with the basics.

The best protein research begins with a great protein assay.

Get back to protein chemistry basics with Pierce BCA (bicinchoninic acid) Protein Assays. The BCA Protein Assay, which was patented by Pierce Biotechnology,1 remains a cornerstone for accurately measuring protein concentration in biological samples.

Quantitate protein concentration from 20-2,000 µg/ml for these applications:

  • Studying protein:protein interactions
  • Measuring column fractions after affinity chromatography
  • Estimating percent recovery of membrane proteins from cell extracts
  • High-throughput screening of fusion proteins

U.S. Patent #4,839,295

Highlights:

  • Colorimetric method; read at 562 nm
  • Compatible with most ionic and nonionic detergents
  • Faster and easier than the Lowry method
  • All reagents stable at room temperature for 2 years
  • Working reagent stable for 24 hours
  • Linear working range for BSA from 20-2,000 µg/ml
  • Minimum detection level of 5 µg/ml with the enhanced protocol
  • Adaptable to microplates
  • Less protein-to-protein variation than dye-binding methods
Comparison of BCA Protein Assay Reagent vs. Lowry Protein Assay
BCA Protein Assay Reagent
1. Mix reagents 1 minute
2. Add sample and incubate 30 minutes
3. Read at 562 nm 1 minute
Total BCA Time: 32 minutes

Lowry Protein Assay
1. Make reagents 70 minutes
2. Add sample and incubate 20 minutes
3. Add Folin Reagent 1 minute
4. Incubate 30 minutes
5. Read at 750 nm 1 minute
Total Lowry Time: 122 minutes

Substance Compatible Conc. Substance Compatible Conc.
NP-40 5.0% Glucose** 10 mM
Emulgen 1.0% EDTA 10 mM
Hepes 100 mM Sodium Chloride 1.0 M
DTT 1 mM NaOH 0.1 M
Guanidine•HCl 4.0 M Ammonium Sulfate 1.5 M
Triton X-100 5.0% Sodium Acetate, pH 5.5 200 mM
Octyl-ß-Glucoside 5.0% SDS 5.0%
Urea 3.0 M Brij-35 5.0%
Sucrose** 40% Lubrol 1.0%
Glycine, pH 2.8 100 mM CHAPS 5.0%

**Always use the same diluent as the sample when constructing a standard curve.
Note: Trichloroacetic acid precipitation of proteins away from these substances results
in better estimation of the protein content in the sample.

References:

  1. Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85.
  2. Sorensen, K. (1992). BioTechniques 12(2), 235-236.
  3. Ju, T., et. al. (2002). J. Biol. Chem. 277, 178-186.
  4. Shibuya, T., et. al. (1989). Tokyo Ika Daigaku Zasshi 47(4), 677-682.
  5. Hinson, D.L. and Webber, R.J. (1988). BioTechniques 6(1), 14, 16, 19.
  6. Akins, R.E. and Tuan, R.S. (1992). BioTechniques 12(4), 496-7, 499.
  7. Tylianakis, P.E., et. al.(1994). Anal. Biochem. 219(2), 335-340.
  8. Gates, R.E. (1991). Anal. Biochem. 196(2), 290-295.
  9. Stich, T.M. (1990). Anal. Biochem. 191, 343-346.
  10. Tuszynski, G.P. and Murphy, A. (1990). Anal. Biochem. 184(1), 189-191

* BCA Protein Assay Reagent A is available in bulk quantities for manufacturing applications.

第二點  兩液相加 前後影響的因素?1985年的論文
第三點  背景值以phosphate buffer saline 鹽分的問題?手冊上說明指出無問題
 
 
吸光採用562的原因 要查原始論文
 
 
法理學的解說 文章修改為一般人可閱讀的形式乎???寄信予助教
 
考古題!
 
 

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