Navigation Links
Total Cellular Protein Determination Using the DC Protein Assay


Introduction
The DC Protein Assay reagents contain both detergent and base which are required for cell lysis of many cell samples prior to determination of total cellular protein. The following protocol can be used for tissue cells; dry cells; protein precipitates from heat, acid, or acetone treatment; or any other samples that require both sodium hydroxide and detergent for solubilization.


Protocol
1. Wash cells or protein precipitate. Using an isotonic solution such as TBS or PBS, suspend the sample in excess buffer and mix by inversion or vortex mixer. Centrifuge sample or precipitate until a tight pellet has formed. Discard the supernatant.

2. Prepare working reagent A' by mixing 20 l Reagent S to each milliliter of Reagent A that will be needed for the assay. Mix by gentle inversion to prevent foaming.

3. Add 1 ml working reagent A' to approximately 1 x 106 cells. Vortex intermittently for 510 minutes, and heat to 60 C (if necessary) to solubilize the pellet.

4. Cool mixture to room temperature. In order to accurately determine protein concentration, several dilutions of the digestion with reagent A' should be made (1:2, 1:4, 1:8, etc.). Record the dilution factors for correction in the final protein concentrations.

5. Create a standard curve. Transfer 100 l of the standard into a clean test tube. Three to five data points should be generated. Either standard supplied with the kit (BSA or IgG) can be used, since both react similarly to the DC Protein Assay reagents.

6. Transfer 500 l sample of serially diluted samples from step 5 into clean test tubes.* To correct for volume difference between samples and standard curve, add 100 l water to each tube. Add 4.0 ml Reagent B and vortex.

7. Add 500 l working reagent A' to samples in the standard curve series and vortex.

8. Add 4.0 ml Reagent B, and vortex.

9. Incubate standards and samples at room temperature for 15 minutes. Measure absorbance at 650750 nm. Compare results to a standard curve.


For technical assistance, call your local Bio-Rad representative, or in the U.S., call 1-800-4BIORAD.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
2. High-Throughput Isolation of Total RNA
3. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
4. Fast Isolation of Total RNA from Small Numbers of Cells
5. Total RNA Isolation Protocol 1,2,3
6. Isolate It All: siRNA miRNA Total RNA Native Protein
7. Isolate Total RNA and Protein From the Same Sample
8. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
9. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
10. Total RNA from Whole Blood for Expression Profiling
11. RNAPrep - Total RNA Isolation Purification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/22/2017)... ... June 21, 2017 , ... ... North Carolina, and engages Timothy Reinhardt to manage the new site. , Tim ... Pfizer Inc, with his most recent role as the Director of Manufacturing and ...
(Date:6/22/2017)... Wendelsheim, Germany (PRWEB) , ... June 22, 2017 ... ... , is taking over the allergy specialists DST Diagnostische Systeme & Technologien GmbH, ... We all know someone who suffers from hay fever, urticaria, asthma, atopic eczema ...
(Date:6/22/2017)... (PRWEB) , ... June 22, 2017 , ... ... media network RegMedNet has produced a Spotlight series on ... scholarly reviews and perspectives by leading experts on the unique regulatory challenges of ...
(Date:6/22/2017)... ... June 22, 2017 , ... The first human cell ... years until the first data on cross-contamination of human cell lines with HeLa cells ... increasing issue in cell culture labs and is associated with dramatic consequences for research. ...
Breaking Biology Technology:
(Date:3/27/2017)... 27, 2017  Catholic Health Services (CHS) has ... Society (HIMSS) Analytics for achieving Stage 6 on ... . In addition, CHS previously earned a place ... an electronic medical record (EMR). "HIMSS ... of EMR usage in an outpatient setting.  This ...
(Date:3/23/2017)... , Mar. 23, 2017 Research and ... System Market Analysis & Trends - Industry Forecast to 2025" ... ... grow at a CAGR of around 8.8% over the next decade ... industry report analyzes the market estimates and forecasts for all the ...
(Date:3/20/2017)... At this year,s CeBIT Chancellor Dr. Angela ... The Chancellor came to the DERMALOG stand together with the Japanese Prime ... partner country. At the largest German biometrics company the two government leaders ... iris recognition as well as DERMALOGĀ“s multi-biometrics system.   ... ...
Breaking Biology News(10 mins):