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:10/11/2017)... ... 11, 2017 , ... Singh Biotechnology today announced that the ... its novel anti-STAT3 (Signal Transducer and Activator of Transcription 3) B VHH13 single ... the cell membrane and bind intracellular STAT3 and inhibit its function. Dysregulation of ...
(Date:10/10/2017)... ... October 10, 2017 , ... ... conjugate (ADC) therapeutics, today confirmed licensing rights that give it exclusive global ... technology developed in collaboration with Children’s Hospital Los Angeles (CHLA). Additionally, ...
(Date:10/10/2017)... ... ... Dr. Bob Harman, founder and CEO of VetStem Biopharma, Inc. spent ... entitled “Stem Cells and Their Regenerative Powers,” was held on August 31st, 2017 ... joined by two human doctors: Peter B. Hanson, M.D., Chief of Orthopedic Surgery, Grossmont ...
(Date:10/10/2017)... 2017 SomaGenics announced the receipt of a ... RealSeq®-SC (Single Cell), expected to be the first commercially ... microRNAs) from single cells using NGS methods. The NIH,s ... accelerate development of approaches to analyze the heterogeneity of ... techniques for measuring levels of mRNAs in individual cells ...
Breaking Biology Technology:
(Date:8/15/2017)... 2017   ivWatch LLC , a medical device company focused ... announced receipt of its ISO 13485 Certification, the global standard for ... for Standardization (ISO®). ... 400 Continuous Monitoring device for the early detection of IV infiltrations. ... "This is an important milestone for ivWatch, ...
(Date:6/23/2017)... ITHACA, N.Y. , June 23, 2017  IBM ... in dairy research, today announced a new collaboration using ... the chances that the global milk supply is impacted ... project, Cornell University has become the newest academic institution ... Chain, a food safety initiative that includes IBM Research, ...
(Date:5/16/2017)... , May 16, 2017  Veratad Technologies, LLC ... of online age and identity verification solutions, announced today ... Identity Conference 2017, May 15 thru May 17, 2017, ... Building and International Trade Center. Identity ... globe and in today,s quickly evolving digital world, defining ...
Breaking Biology News(10 mins):