Dissolve 100 mg of CBB G-250 in 50 ml of ethanol, add 100 ml of 85% phosphoric acid and make volume to 1lt with distilled water
The Bradford reagent is prepared as follows:
1. Dissolve 100mg Coomassie Brilliant Blue G-250 in 50ml 95% ethanol, add 100ml 85% (w/v) phosphoric acid.
2. Once the dye has completely dissolved, dilute to 1 litre with deionised water.
3. Filter through Whatman #1 paper just before use.
Preparation for 1000ml Bradford reagent:
1. Dissolve 100mg Coomassie Brilliant Blue G-250 in 47ml Methanol.
2. Once the dye has been completely dissolved, add 100ml 85% (w/v) Phosphoric Acid.
3. Filter it through Whatman filter-paper # 1.
4. Make up volume up to 1000ml with De-ionized Water.
5. Store it in Amber Glass Bottle at 4°C.
6. Use this prepared reagent within 6 months.
Does anyone know which method gives better results between using methanol and ethanol to prepare Bradford reagent?
Yogesh Chandra Tripathi Sonam Sneha Saria Tariq
Ethanol or methanol, either of the two can be used as the role of both methanol and ethanol in the Bradford reagent is to act as a reacting functional group, since the OH at the end of the ethanol/methanol can be depronated. Both the solvents help prevent aggregation of proteins during the assay.
Bhavya G. Yogesh Chandra Tripathi Thank you so much both of you.
Wipa
The quality of ortho-phosphoric acid greatly affects the quality of the reagent. Acid is more important than alcohol. Ortho-phosphoric acid changes over time.
That is the second time that I read that Ethanol an Methanol can be deprotonated. Bradford uses ~13% phosphoric acid (w/w), how is it relevant that the alcohols can be deprotonated? As far as I know it is not required for a chemical reaction but just for dissolving the CBB.
In my hands I found that decreasing the CBB concentration from 100mg/L in the original recipy, to 70-80 mg/L is improving the staining and reducing the background.
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