I did not less than 10 times of Bradford assay, but unfortunately I still be having bubbles in my samples or some BSA standards (although I'm getting lesser bubbles compared to previous).
I found an website and it mentioned that "If bubbles are present, use a P20 to add 5 microliters of ethanol to the surface of the well -- this breaks bubbles without affecting the absorbance."
I try to find the reason behind why ethanol wont affect the absorbance but I cannot find it.
Do anyone know the theory behind?
Hi! The principle behind Bradford assay is that the dye binds to non-polar aminoacids, and is quite robust on a range of conditions, only higher SDS concentrations can affect the assay, lower ethanol concentrations will not, since it doesn't affect pH or hydrophobicity (important factors for the Bradford assay). You should probably worry about getting bubbles in your sample because that leads to denatured proteins. Try to pipet slowly and against the walls of the tube/plate, avoid generating drops.
Cheers!
Another way to pop bubbles on the surface of the liquid in the wells is to blow methanol or ethanol vapor over them. This is done by putting a small amount of alcohol in a laboratory squeeze bottle, the kind with a removable tube that attaches to the cap. The tube is removed so that only the vapor comes out when the bottle is squeezed. Acetone probably works, too.
You get bubbles when some of the liquid in the pipet tip sticks to the inside of the tip, so that air blows out when you expel the liquid. This problem can be reduced by pre-wetting the tip with the liquid to be dispensed by drawing it up and expelling it a few times first before pipetting into the plate.
I think the ethanol will affect the absorbance, because you change the volume in your cuvette/well. You have a dilution and if you don't add in everey sample ethanol but only in the one with bubbles, you have bad results.
What I do is to disturb the bubbles with a needle.
Thanks for the answers guy!!!!
Nicolas, how does bubble denature proteins? I have try to pipet my BSA and my proteins slowly and yet I still get the bubbles at some wells.
Adam, it's a better idea than adding 5 microlitters of ethanol into the well with bubbles as I afraid it will affect the absorbance. How to pre-wet the tip? I got bubbles when I pipetted the BSA and my protein samples
Stefanie, this is what I am worrying about. I read the microplate without adding ethanol to each well (just particular well)
We had the experience that with small bubbles on the wall make no trouble, because the reader is measuring in the middle. Maybe You can try a BSA solution with in one well a lot, another less bubbles and see the differences.
If you're worried about it, try multiple controls using duplicates of BSA standards +/- ethanol to demonstrate to yourself that ethanol isn't affecting the numbers. As Stefanie noted, if you do add it to your experimental samples, you'll need to add it to all samples and standards.
By pre-wetting the pipet tip, I mean that you draw up the sample, then expel it back into the container, then draw it up again for dispensing. The tip will be coated by the solution the first time. This procedure should always be used with protein and detergent solutions that stick to the tip.
If you have electronic pipettors, you can avoid the necessity of pre-wetting because the pipettor can be set up to draw in more volume than it dispenses.
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