Making 2% skimmed milk in PBS
Alex Chang
I am trying to make 2% skimmed milk in PBS for blocking during my panning
procedures. I am wondering how to make it sterile. I know you can
autoclave it. I did it last time, the bottle returned to me with brown
solutions, I guess it was overdone. Well, our autoclaving is done by a
centralized service. That means we don't have control over the time and
temperture. I also tried filtration using 0.2 um filters, but the milk
solution clogged the filter easily.
Any solutions to this problem?
--
Alex Chang
Pathology
University of British Columbia
ach...@hivnet.ubc.ca
Mick Partis
I guess that autoclaving any protein solution is going to denature it
whatever the duration. I would suggest that you use a purified casein
and filter sterilise. To get casein into solution, you will need to heat
to 50-60 C, or dissolve in a very alkaline buffer
--
mick....@hri.ac.uk Horticulture Research International
http://www.geocities.com/CapeCanaveral/1957/
Steve
If you were just worried about bacteria growing, you could make your
solution as sterilely and quick as possible, and then add .02% sodium
azide. Nothing will grow in that. We never sterilize our blocking
buffers, and have never had a problem...
Steve
Bowman Gray School of Medicine
W-S, NC, 27157
Jan Hoolwerf
Hello,
lp...@unixg.ubc.ca (Alex Chang) wrote:
AC> I am trying to make 2% skimmed milk in PBS for blocking during my
AC> panning procedures.
That should be possible. As far as i know some manufacturers sell a product
that is made from skimmed milk but purified in some way.
AC> I am wondering how to make it sterile.
Is that necessary? Don't think so. Make the solution fresh before use from a
good quality milk powder and i doubt you will have problems with growth of
bacteria.
We produce a skimmed milk powder for a test procedure in the dairy industry and
milk from this powder is used without sterilisation and at 30 øC for six hours.
AC> I know you can autoclave it. I did it last time, the bottle returned
AC> to me with brown solutions, I guess it was overdone.
Not overdone, it's the Maillard reaction. Protein with the lactose.
As an alternative you can pasteurize it 15 min. at 100 øC. But it still will be
light brown.
AC> I also tried filtration using 0.2 um filters, but the milk solution
AC> clogged the filter easily.
I know. You cannot filter milk through such tine holes <s> and milk from powder
is even worse.
with regards, Jan Hoolwerf
******************* these words were mine, all mine !! ********************
J.D. Hoolwerf
Neth. Inst. for Dairy Research
P.O. Box 20 phone : 31-318-659511
6710 BA Ede fax : 31-318-650400
the Netherlands e-mail : hool...@solair1.inter.nl.net
***************************************************************************
Lazrs Lng
Try using a .45 micron filter first, with one of the prefilters in the
setup . . . and then if it works, filter again through the .22 . . .
Lazarus Long
marcel hoffmann
Alex Chang wrote:
>
> I am trying to make 2% skimmed milk in PBS for blocking during my panning
> procedures. I am wondering how to make it sterile. I know you can
>
Dear Alex,
Try adding 0.3% sodium azide to the milk that will keep it
free of any growth for a while.
--
********************************************
Marcel Hoffmann Ph.D.
Dept. of Pharmacochemistry
Vrije Universiteit Amsterdam
de Boelelaan 1083, 1081 HV Amsterdam
tel: +31(0)20-4447572
fax +31(0)20-4447610
********************************************
Robert Willows
We routinely use 15mM Sodium Azide in our 2% skim milk to stop bacterial
growth. Although, I don't think you can do this if you use a HRP
conjugated secondary because I think the azide is inhibitory.
--
Robert Willows
Email: Robert_...@brown.edu
Richard P. Grant
In article <Robert_Willows-...@128.148.57.20>, Robert_...@brown.edu (Robert Willows) writes:
> We routinely use 15mM Sodium Azide in our 2% skim milk to stop bacterial
> growth. Although, I don't think you can do this if you use a HRP
> conjugated secondary because I think the azide is inhibitory.
If you wash your filters properly, it's OK.
Azide is not necesssary for a 60' block.
R
--
Richard P. Grant MA DPhil rpg...@molbiol.ox.ac.uk
Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
http://users.ox.ac.uk/~lady0266
No wonder I can't go to parties anymore.
P.F. Watson
On the question of filtration in general.. it usually helps to centrifuge
the solution first to remove those stubborn iddy biddy bits that never seem
to go. I guess you might lose 0.001% of the powder you weighed out though..
zzzzzzzzzzzz
martin LEACH
Alex Chang (lp...@unixg.ubc.ca) wrote:
: I am trying to make 2% skimmed milk in PBS for blocking during my panning
: procedures. I am wondering how to make it sterile. I know you can
: solutions, I guess it was overdone. Well, our autoclaving is done by a
: solution clogged the filter easily.
: Any solutions to this problem?
did you try using several pre-filters in front of the 0.2 micron filter...
this works well for us when filtering chick embryo extract etc... and other
assorted goos....
later
Martin
: --
: Alex Chang
: Pathology
: University of British Columbia
: ach...@hivnet.ubc.ca
--
..... Martin Leach Email:le...@bu.edu
_|____ Dept. of Pharmacology Phone: (617) 638-5323
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Technical Assistance
Robert Willows wrote:
>
> We routinely use 15mM Sodium Azide in our 2% skim milk to stop bacterial
> growth. Although, I don't think you can do this if you use a HRP
> conjugated secondary because I think the azide is inhibitory.
>
Azide is indeed an inhitor of HRP activity. However, at such low concentrations,
it usually is not a problem. An alternative would be to add thimerasol.
Alix Helme-Guizon
On Thu, 3 Oct 1996, Robert Willows wrote:
> We routinely use 15mM Sodium Azide in our 2% skim milk to stop bacterial
> growth. Although, I don't think you can do this if you use a HRP
> conjugated secondary because I think the azide is inhibitory.
>
sodium azide is very efficient against bacterial growth.
i use only 0.00001% to keep sterile
my 10X buffer. After dilution and addition of milk (arbitrary amount,
about 2 spouns for 50ml, this doesn't matter), only trace of azide
will remain. I've never seen inhibition of my secondary antibody (ECL
chemiluminescence detection) by doing so. Neither did i see any bacteria
in my buffers!
i didn't try do stock milk solutions in that way but you can try.
regards,
Alix