SDS quantitation and removal with resins

Dr Engelbert Buxbaum wrote:

Hi,

THanks for the info. I think the >CMC fluorescence method is as close as
I'm going to get to an assay method... unfortunately, I dont think it's
going to be usefull for me.

I agree that the hydrophobic bead method may be the best bet for
removal. I knew about the pierce product already (if you mean the
SDS-Out product), and had wondered what they use in that. They say it's
a 'precipiation reagent'... but the spec sheet and instruction 'manual'
on their website doesnt really give any information about whats in it or
how it works. Have you used it before? You say that it's column-based;
does it use hydrophobic beads in that or do they rely on a (at leatst
semi-) specific precipitation of SDS?

Anyhow, thanks for the info.

Could the Pierce reagent simply be a solution of KCl or KAc?

Potassium dodecyl sulfate is fairly insoluble in water. Thus one can remove
most of the "SDS" from a solution simply by adding ~0.75 M K+ to a sample.
I do not have a clue what the final concentration of detergent will be... it
will vary greatly with other components present. But it might work in your
application.

As for using various resins... all have the potential problem of absorbtion
of the protein of interest to the resin. This is not usually an
insurmonttable problem, just beware. (I have had good luck in reducing loss
of protein when removing non-ionic detergents using "Biobeads" by adding
"extra" lipid to the mix. Not an ideal fix, but one does what one has to!)

As for quantitation of detergents... not easy! The fluorescence assays,
while they work well to measure CMC in a simply solution are not specific
enough for a sample containing biological molecules.

In the "old days" (late 70's), one could by radiolabeled SDS and TX-100 from
NEN. (I don't know if they are still available.) This was the simplest way
to follow the removal of detergent... add a 1000 cpm / uL of 3H-detergent to
the starting sample and count small samples along the way.

--- FRG

Frank R. Gorga, Dept. of Chemical Sciences, Bridgewater State College


"Scott Coutts" <[Only registered users see links. ].au> wrote in message
news:[Only registered users see links. ].au...

Hi Frank,

Frank R. Gorga wrote:


Yeah, some others have suggested that, and that's what I had suspected.
But we dotn have any, and I've never used it! So I was wondering if
anybody knew for sure.


Yeah, I have used biobeads and an amberlite xad with quite a lot of
success, but that was with Triton X-114. I didnt know if it worked with
SDS as well.


No, I had expected that it wouldnt be good enough for what I want but I
think there isnt really a method that is.


Yeah, I know you can buy radiolabelled SDS from Sigma, but I cant really
do that. It has to be 'normal' SDS.

Thanks anyhow.

Scott Coutts wrote:



It may not give you absolute measurements in a complex sample, but
semiquantitative determinations should help to judge efficiency of
detergent removal.



The stuff we used a couple of years ago came prepacked in disposable
columns, and worked on the hydrophobic bead principle, and was supposed
to work with all detergents. There may be different products available
though, I don't have a catalogue handy.

FWIW I prefere to add beads to thee sample manually, losses of protein
are lower that way and its cheaper too.

Hello Folks,

I am trying to purify ATP (commercially supplied - Sigma/
Calbiochem) from its hydrolysis product ADP (even 3-5% ADP contamination
is not acceptable for my experiments).

Running a KCl gradient on an ion-exchange column separates ADP
from ATP. However, the ATP gets very diluted coming off the column.
I cannot increase the stock ATP that I load on the column, because the ADP
and ATP peaks are closely spaced and at high ATP concentrations, the two
peaks begin to merge.

Moreover, the ATP elutes at about 50-100 mM KCl.

My main problem is to remove the KCl from the ATP solution without
enhancing ATP hydrolysis in any manner AND also concentrate the ATP soln.
to obtain a greater working concentration (although the latter is not
absolutely essential).

changing pH conditions, temperature, lyophilization, etc., could
promote hydrolysis of ATP to give ADP, which defeats the purpose of my
purification procedure.

either I am missing something very obvious, or it is indeed
difficult to do this!

Any useful and tested insights on how to purify and concentrate
ATP without promoting hydrolysis will be of great help.

Thanks.

Best wishes

Bhadresh Rami

"Bhadresh Rami" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

May I ask you what is the downstream application the purified ATP is going
to be used in? If it is some sort of enzymatic reaction and ADP in the
sample is your concern, then you could use an ATP regeneration system to
remove convert ADP to ATP. One good example is creatine phosphokinase and
phosphocreatine.
-Emir

Bhadresh Rami wrote:


I have once dabbled with ion pairing chromatography of nucleotides on
C18-columns, using 0-1 M diethylamin bicarbonat as buffer. This buffer
is volatile and can be removed by lyophilisation. If the gradient slope
is adjusted properly, ATP, ADP and AMP/Pi can be base-line separated in
under 15 min.

The problem with this buffer is that it is difficult to get the pH below
7.5, which is required to keep the silica backbone in the column from
dissolving. Soda-Stream soda-water generators from your local food store
sort of work.

In theory, resin-based C18 columns would solve the problem, as they are
not pH sensitive. However, I did not have the money to try this out.

I have also tried diethylamine formiate and acetate buffers for ion
exchange and ion pairing, but found that these acids are far less
volatile than the diethylamine. During lyophilisation you end up with
your product in concentrated acetic of formic acid, with predictable
consequences.