Navigation Links
Modification of Bio-Rad DC Protein Assay for Use With Thiols


The following protocol is an adaptation for protein determination in the presence of reducing agents such as DTT (dithiothreitol) and BME (2-mercaptoethanol). This adaptation will effectively neutralize up to 50 mM BME or 5 mM DTT.


Materials Required
DC Protein Assay, Bio-Rad Laboratories catalog numbers 500-0116, 500-0111, 500-0112

Iodoacetamide

DTT, 1 g, Bio-Rad Laboratories catalog number 161-0610

2-mercaptoethanol, 25 ml, Bio-Rad Laboratories catalog number 161-0710


Instructions for Standard Assay
1. Prepare a solution of 0.1 M iodoacetamide in 0.1 mM Tris buffer, pH 8.0.

2. Prepare working reagent A' for DC Protein Assay by adding 20 l of Reagent S to each ml of Reagent A that will be required for the run. (This working reagent A' is stable for 1 week even though a precipitate may form after 1 day. If a precipitate forms, vortex. Do not pipet the undissolved precipitate, as this will likely plug the tip of the pipet, thereby altering the volume of reagent that is added to the sample.)

3. Prepare 3 to 5 dilutions of a protein standard containing 0.1 to 2.0 mg/ml protein in a buffer containing approximately the same concentration of DTT or BME as the sample. A standard curve should be prepared each time the assay is performed. For best results, the standard should be prepared in the same buffer as the sample.

4. Pipet 100 l of standards and samples into clean, dry test tubes.

5. For samples containing BME, add 100 l iodoacetamide to each standard and sample test tube. For samples containing DTT, and 200 l iodoacetamide to each standard and test tube. Vortex samples and incubate at 37 C for 15 minutes.

6. Cool for 5 minutes at room temperature.

7. Add 500 l of reagent A' into each test tube. Vortex.

8. Add 4.0 ml of Reagent B into each tube and vortex immediately.

9. After 15 minutes, measure the absorbance of each standard and sample at 750 nm. The absorbances will be stable for at least 1 hour.


Instruction for Microtiter Plate Assay
Scale down volumes proportionately.


Reference
Analytical Biochemistry, 170, 203208 (1988).

back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. The Bio-Rad Silver Stain, Rev E
2. A New C-Terminal GST Vector for Protein Production in S. pombe
3. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
4. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
5. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
6. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
7. Optimized Imaging of Protein Gels Stained with Coomassie Brilliant Blue Dye
8. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
9. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
10. Yeast Protein Production System Features High Yields and One-Step Purification
11. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/27/2017)... ... March 27, 2017 , ... A research team led by ... in JoVE’s Video Journal, the world’s first peer-reviewed scientific video journal. The article ... coronary artery disease (CAD). Lam is an assistant professor at the Department of ...
(Date:3/24/2017)... and ROCKVILLE, Md. , March 24, ... Maxwell Biotech Venture Fund (MBVF), today announced positive results ... to the standard drug therapy regimen in patients with ... molecule drug discovered by scientists at Sequella, Inc. ( ... of Health. A total of 140 ...
(Date:3/24/2017)... Agenus Inc. (NASDAQ: AGEN), an immuno-oncology company ... vaccines, today announced participation at the following conferences: ... and Maidstone Life Sciences conference "Cancer Immunotherapy Conference" at ... New York, NY . Agenus will participate in ... am: Robert B. Stein , M.D., Ph.D., President, ...
(Date:3/23/2017)... MILFORD, Mass. , March 23, 2017 ... leading partner to global in vitro diagnostics ... launch of the industry,s first multiplexed ... inherited disease testing by next-generation sequencing ... materials were developed with input from industry ...
Breaking Biology Technology:
(Date:3/24/2017)... 24, 2017 Research and Markets has announced ... Analysis & Trends - Industry Forecast to 2025" report to ... The ... a CAGR of around 15.1% over the next decade to reach ... analyzes the market estimates and forecasts for all the given segments ...
(Date:3/23/2017)... 23, 2017 Research and Markets has announced ... & Trends - Industry Forecast to 2025" report to their ... The ... CAGR of around 8.8% over the next decade to reach approximately ... the market estimates and forecasts for all the given segments on ...
(Date:3/22/2017)... , March 21, 2017 Optimove ... used by retailers such as 1-800-Flowers and AdoreMe, ... — Product Recommendations and Replenishment. Using Optimove,s machine learning ... personalized product and replenishment recommendations to their customers ... on predictions of customer intent drawn from a ...
Breaking Biology News(10 mins):