wavelenght for Bradford - (Oct/18/2006 )
hi,
In my lab we have a spectrofotometer which reads at a maximum of 505 nm, but I´ve been told that to perform a bradford assay you must read at 595 nm. If I read at 505 nm would I obtain a very wrong result? 
thanks 
I didn't try , but I would not recommend.
the absorption shifts from 465 to 595 nm while binding to proteins. So, with 505 you are really close to 465.
Thanks little mouse 
The available wavelenghts in my spectofotometer are 405, 450, 492 and 550 nm (it was bought for ELISAs). I have the Bradford assay from Biorad, and the recommended OD is 595 nm. Reading at 505, am I underestimating a lot my protein concentration? 
And another question: Before reading, I have to choose a "reading wavelenght" and also a "reference wavelength", what does this mean? What should I do (apart from talk to my boss and convince him to buy the appropiate spectro)? 
thanks
The available wavelenghts in my spectofotometer are 405, 450, 492 and 550 nm (it was bought for ELISAs). I have the Bradford assay from Biorad, and the recommended OD is 595 nm. Reading at 505, am I underestimating a lot my protein concentration?
And another question: Before reading, I have to choose a "reading wavelenght" and also a "reference wavelength", what does this mean? What should I do (apart from talk to my boss and convince him to buy the appropiate spectro)?
thanks
about reference wavelength, when you do ELISA with AP, you read at 405nm, and use 620 nm (if I remember well) as reference. It's an internal control.
why don't you ask in your neighbourg labs if they have the right spectrophotometer? I don't know how it is in your insititute, but where I used to work, we often go in other labs to do some little things like that. It's no use that every lab buy all the apparatus.
If you can read on 595nm I would then read at 505 and check, but I bet it will not be OK.
Yes, I think I will ask to the other people, it´s a common apparatus, possibly they have one.
Thanks little mouse
Thanks little mouse
not to recommend as most discuss; use alternative wavelengths for different methods:
205 nm (directly without dye)
280 nm (directly without dye)
750 nm (modified Lowry assay)
If I had no other option than bradford and a spec that doesnt go up to 500 nm .... I will make the reading anyway, of course using different standards at different concentrations. You will detect at 500 nm at least part of the peak coming from the absorbance at 600 nm.
But I agree with kosmodrom
lots of thanks everybody
Ok , I did a bradford, and just to confirm what i said I tried to read at 490 nm, and it doesn't work at all as expected.
by the way, I also tried 650 nm (our 570 is no more working), it works, but the signal is not as strong, and this leads to a less good resolution of the assay.
Oh, thanks missele.