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Bradford assay - protein quantification using BSA Standard. but i encounter problems

Bradford assay - protein quantification using BSA Standard. but i encounter problems - Protocols and Methods Forum

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Old 04-24-2008, 01:21 AM
Pipette Filler
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Default Bradford assay - protein quantification using BSA Standard. but i encounter problems



hi i'm new here. soorrry if i make any mistake.

i am using Bradford Assay to quantify protein and BSA as my standard.
1) I prepared a master mix of 2mg/mL of BSA and diluted them from 0.1mg/mL to 1.2mg/mL in a linear range.
2) thereafter i diluted the bradford reagent at 2:7, adding 4mL of reagent to 14mL of ddH2O.
3) I added 20uL of each BSA standard into 980uL of the diluted bradford reagent, prepared in step 2.
4) samples were incubated for 15 minutes and OD595 were gathered to plot a curve.

Problem: all values i obtained is lower than 0.03. what do u think the problem is?
UV curvette and GV curvette. what's the difference? which should i use.
however i also use nanodrop, the value still remain as BAD
thanks alot in advance =)
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Old 10-22-2008, 02:29 AM
Edi Edi is offline
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Default Re: Bradford assay - protein quantification using BSA Standard. but i encounter probl

Your incubation time is too long. Usually 5 minutes incubation is enough.
Also, check whether you have detergent contamination - detergents interfere with the assay.
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Old 10-22-2008, 02:17 PM
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Thumbs down Re: Bradford assay - protein quantification using BSA Standard. but i encounter probl

zz. noob! up!.

if too long. value shld be very high isn't it.
or at least the color shld change alot, including the blank.
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