protein concentration - bradford method (Jan/20/2007 )
Hi there,
I would like to know if you measure a protein concetration the same way that I do. Before I used to use the Bradford method with the dilutions using BSA but I do not do that anymore. Actually I dilute 1ml of Biorad in 4ml of water, I transfer 1ml of this dilution in a cuve and then I add 1ul of protein, but the point is I took out this 1ul from 100ul of the total volume of my elution fractions (around 3ml). I obtained a very low conecentration when I measured the absorbance using the method already set up by the spectrophotometer, which is surprising regarding to the gel I got after purification. Should I take 1ul of the total volume of the elution fractions? (I am a bit afraid of doing that because I've already frozen the aliquots of protein, however I could get the right concentration).
well i do as you (800µl water, 1µl sample and 200µl solution) but get nice results.
If your OD is too low, use 2-5 µl (not really affecting your dilution factor) and see
I would recommend to quantify your fractions just after eluting to see how much you get in an aliquot.
If your OD is too low, use 2-5 µl (not really affecting your dilution factor) and see
I would recommend to quantify your fractions just after eluting to see how much you get in an aliquot.
Thanks for your reply. I actually tried to use 2ul and the concentration got a bit higher but I think it is not a precised measurement
. If your OD is too low, use 2-5 µl (not really affecting your dilution factor) and see
I would recommend to quantify your fractions just after eluting to see how much you get in an aliquot.
Thanks for your reply. I actually tried to use 2ul and the concentration got a bit higher but I think it is not a precised measurement
.Bradford is never a precise measurement, in addition, the pipetting error result from handling 1 -5 ul would also be the main cause of error in bradford test......up to me, 5 - 8% error can be accepted when I used bradford
I would like to know if you measure a protein concetration the same way that I do. Before I used to use the Bradford method with the dilutions using BSA but I do not do that anymore. Actually I dilute 1ml of Biorad in 4ml of water, I transfer 1ml of this dilution in a cuve and then I add 1ul of protein, but the point is I took out this 1ul from 100ul of the total volume of my elution fractions (around 3ml). I obtained a very low conecentration when I measured the absorbance using the method already set up by the spectrophotometer, which is surprising regarding to the gel I got after purification. Should I take 1ul of the total volume of the elution fractions? (I am a bit afraid of doing that because I've already frozen the aliquots of protein, however I could get the right concentration).
BSA as the reference protein is not ideal as it has more hydrophobic sites than an average protei ; use a standard mixture of various proteins, or if using a single protein: gamma-globulin or lysozyme
Actually, my lab has just invested in a new machine called nanosomething, I don't remember the name, anyway. It's quite simple: you take 2µl of your dialysis buffer as the reference and then take 2µl of your protein and you get the concentration. The problem is I throw out the buffer that I used when I dialysed my protein . So should I used a fresh dialysis buffer as the reference or should I repurify the protein to dialyse it again?
Regards.
. So should I used a fresh dialysis buffer as the reference or should I repurify the protein to dialyse it again?Regards.
you can probably get away with fresh buffer if you don't mind a little error. if you need to be accurate then you should redialyze your protein. me? i would just use fresh buffer.
. So should I used a fresh dialysis buffer as the reference or should I repurify the protein to dialyse it again?Regards.
you can probably get away with fresh buffer if you don't mind a little error. if you need to be accurate then you should redialyze your protein. me? i would just use fresh buffer.
But if you want precise exp and you have sufficient amount of protein and if you have centricon tubes suitable cutoff you may concentrate your sample ( 10000g 5-10 min) and pick up flow through fraction - it is your protein dialysis buffer. But essentially I agree with mdfenko about using fresh buffer. Don't lose yuor time, protein and money!
I would like to know if you measure a protein concetration the same way that I do. Before I used to use the Bradford method with the dilutions using BSA but I do not do that anymore. Actually I dilute 1ml of Biorad in 4ml of water, I transfer 1ml of this dilution in a cuve and then I add 1ul of protein, but the point is I took out this 1ul from 100ul of the total volume of my elution fractions (around 3ml). I obtained a very low conecentration when I measured the absorbance using the method already set up by the spectrophotometer, which is surprising regarding to the gel I got after purification. Should I take 1ul of the total volume of the elution fractions? (I am a bit afraid of doing that because I've already frozen the aliquots of protein, however I could get the right concentration).
Unless you did something different to the remaining elutions that you now have frozen, that 1ul is the same no matter if it came from an aliquot or from the entire sample.
Ok, thanks everyone for your suggestions. I have used the fresh buffer and the concentration seems to be reasonable.