Hi Everyone,
Thank you so much for your additional tips about various kits. My protein
is tagged and when I cleave off the tag (a step that needs reducing agent),
I will unfortunately have both detergent and reducing agent in my protein
buffer.
Nicolas, thanks for your word of caution. I can't th
No, because Bradford is based on the increase in absorbance when the dye moves
from a hydrophilic environment to a hydrophobic one (like the protein interior,
or like the interior of a micelle). When detergents are present in excess of
their CMC, the change in absorbance from partitioning into t
AC.UK] de la part de Niks
[nik...@gmail.com]
Envoyé : vendredi 14 février 2014 07:45
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] Determining concentration of membrane protein
Dear All,
May be a stupid question. But if we take buffer with detergent as control
(Blank), would not the difference i
x)
Nicolas
De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ho Leung Ng
[h...@hawaii.edu]
Envoyé : vendredi 14 février 2014 01:58
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] Determining concentration of membrane protein
Hi Raji,
There are
Dear All,
May be a stupid question. But if we take buffer with detergent as control
(Blank), would not the difference in ODs using any of the methods used e.g.
Bradford assay, gives protein concentration?
Regards
Nishant
On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett wrote:
> Your basic choic
Hi Raji,
There are also some proprietary stains such as the "660 nm" (can't
they think of a better product name?) stain from Pierce that are detergent
compatible. I used this briefly with success when comparing against Abs 280
nm.
Ho
Ho Leung Ng
University of Hawaii at Manoa
Assistant Prof
Hi Everyone,
Thanks very much for your helpful responses and suggestions. I will use the
BCA assay.
Cheers,
Raji
On Thu, Feb 13, 2014 at 11:27 AM, R. M. Garavito wrote:
> Roger,
>
> While I agree with your list, the BCA assay does not use molybdate (as we
> make it from scratch with bicinchoni
Roger,
While I agree with your list, the BCA assay does not use molybdate (as we make
it from scratch with bicinchoninic acid, sodium carbonate, sodium bicarbonate,
sodium tartrate, and cupric sulfate pentahydrate). For membrane proteins, I
prefer the BCA assay until the protein is pure enough
Your basic choices for protein assays are:
1. Alkaline copper methods (e.g., Biuret and micro-biuret)
2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
3. Hydrophobic dye methods (e.g. Bradford)
4. UV methods (e.g., A280, A230, A210, etc.)
Method 1 is least sensitive to amino acid
I think that BCA assay is what you are looking for.
Le 13 févr. 2014 à 16:06, Raji Edayathumangalam a écrit :
> Dear CC4BBers,
>
> I am trying to figure out what is the best way to determine the protein
> concentration of my membrane protein. My purified membrane protein is in 20mM
> Tris pH
Dear CC4BBers,
I am trying to figure out what is the best way to determine the protein
concentration of my membrane protein. My purified membrane protein is in
20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
After reading the friendly manuals and searching online, I've learned that
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