User:Cedar J. Fowler/Notebook/Western Blot

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Project Description/Abstract

 * BRADFORD ASSAY

Materials

• Protein assay dye 5x (BioRad) • 2ml microcentrifuge tubes • 96 well plate flatebottom plate • Plate reader • Distilled H2O • 2mg/mL BSA

Protein Assay (Bradford)

1. In 96 well plate, carefully pipette 200μL protein assay dye 1x (BioRad) in each well. Protein assay comes as 5x concentrate, dilute with distilled H20.

2. Standard: Prepare 7 standards via serial dilution. Starting with 30uL distilled H2O and 30μL BSA standard [2mg/mL]. To each remaining standard, add 30 μL of the previous sample. The final sample will have 60μL

3. Sample: To 27 μL Distilled H2O, add 3 μL sample. This creates a 10x dilution. If sample is suspected to be very concentrated, test another dilution of 3uL in 57uL distilled H2O. This creates a 10x dilution.

4. Add 10μL sample to wells in duplicate, mix well by pipette.

5. Incubate at room temperature ~5 min.

6. Running the sample: Choose old plate as template and modify to fit current samples. Ensure that Standards and Unknown concentrations are correct (Standard is Serial dilution, 2x, X1 is most concentrated; Unknown is 10x dilution or 20x dilution) Save as a new file. Open Endpoint program and pick presaved plate file. Change reports to include raw data and standards. Change wavelength to read A593.

7. Export Concentrations to excel, remember to start export at cell 1, add sample IDs, and email to self.