Maleic acid buffer: Help me make it
Scott C. Johnson
things on me a bit. Namely, one of the new buffers calls for 100mM Maleic
acid, 150mM NaCl, pH 7.5. Seems easy right. Well, the 1x buffer is, only
problem is that you need alot of it, and a 10x stock would be much more
convenient. B-M sells a 10x stock, but there's not much sense in buying
the buffer, when it's easy to make, or so I thought. Before I describe the
problem, I'll tell you that I've been on the line with B-M tech services
several times, and they cannot solve it. Although they can make the
buffer.
So, basically the 10x sotck requires 1M Maleic acid, right now let's
forget about the NaCl, actually we'll never even get to it. We'll make one
liter, and since the FW of Maleic acid is 116, we'll use 116 grams. Stir
it up, and in it goes. So now we have about 800 mls of water with 116
grams of Maleic acid completely disolved. Next the recipe calls for pHing
with NaOH pellets or concentrated solution. I've tried both, and get the
same result. The pH of the acid solution is about 1.3, and we've got to
get it to 7.5. I start adding NaOH, and before the pH hits 2.0, all the
Maleic acid starts falling out, crystalizing like crazy. I've tried heat,
and tons of stirring, but that stuff is not going back in.
Well, that's about all you need to know. If you know how to suceffully
make a 1M stock of Maleic acid pH 7.5 I would appreciate you sharing. B-M
says they use the same stuff I did, the free acid FW 116. I even ordered
new Maleic acid, as ours was old. As would be expected. Both the old Sigma
stuff and the new VWR stuff did the same thing. So the tech services guys
said to try a 5x solution. Not really a reasonable explanation, but I
tried, and even 500mM Maleic acid precipitates at pH 2.0.
Am I an idiot? What am I missing? What are they doing differently?
Thanks,
Scott
--
Scott C. Johnson
AHABS
Univ. of Wisc. - Madison
s...@calshp.cals.wisc.edu
Nikolai Chitaev
> Subject: Maleic acid buffer: Help me make it
> Date: 15 Feb 1996 15:09:12 GMT
>
<snipp>
>
> Am I an idiot? What am I missing? What are they doing differently?
>
> Thanks,
> Scott
>
Hi, Scott.
No! You a no more idiot than people at BM. You only miss one thing. They,
i.e. people at BM, had never ever made 1M maleic acid buffer, pH 7.5.
If you`ll read carefuly p80 of BM catalogue (1996) "Genius Wash and Block Buffer Set"
you`ll see that products they sell are 10x solutions in respect to anything, but are
1x solution in respect to maleic acid buffer. They even sell 1x buffer to dilute their
10x stock solutions!
So, do ahead, prepare all solution yourself using 0.1 M maleic acid, pH 7.5, but 10x with
respect to other components, and then additionaly preparte a lot of 0.1 M maleic
acid, pH 7.5 to dilute those 10x solutions.
I can assure you that 0.1 M maleic acid solution with pH 7.5 could be easy prepared.
I did it. Essentialy as described.
Nikolai
http://128.252.119.253
Rich Kernan
s...@calshp.cals.wisc.edu (Scott C. Johnson) wrote:
> I have long used the Genius detection system, however, B-M has changed
> things on me a bit. Namely, one of the new buffers calls for 100mM Maleic
> acid, 150mM NaCl, pH 7.5. Seems easy right. Well, the 1x buffer is, only
> problem is that you need alot of it, and a 10x stock would be much more
> convenient. B-M sells a 10x stock, but there's not much sense in buying
> the buffer, when it's easy to make, or so I thought. Before I describe the
> problem, I'll tell you that I've been on the line with B-M tech services
> several times, and they cannot solve it. Although they can make the
> buffer.
>
> (rest deleted)
>
> Thanks,
> Scott
>
> --
> Scott C. Johnson
> AHABS
> Univ. of Wisc. - Madison
> s...@calshp.cals.wisc.edu
Scott,
Heh, I had the same problem, and no one in my lab was able to make 1M
maleic at pH 7.5. What we do is mix up a stock of 1M maleic. The pH
turns out to be around 1. We use this as a "stock" and make large
quantities of 1X maleic acid buffer by adding stock 5M NaCl, pH-ing and
then bringing up the volume to reach the appropriate concentrations. Not
as nice as a 10X buffer, but not as frustrating as repeated failure. I
have no idea how BM is able to make a 10X solution.
--
Rich Kernan
Dept of Microbiology
University of GA
DOUGLAS PRASHER
Have you tried adding NaOH to get the pH significantly beyond 2?
Douglas Prasher
Jon
>Nikolai
>http://128.252.119.253
After calling B-M's tech support, I found out the same thing. A 10 X
solution is 0.1M
Jon Cowan
Dept. of Biology
University of Alabama at Birmingham
jco...@earthlink.net
Rosanne Casu
>In article <scj-150296...@jmm3.ahabs.wisc.edu>,
>s...@calshp.cals.wisc.edu (Scott C. Johnson) wrote:
>>That there were problems making up a 10x stock of buffer 1 i.e. 1M maleic acid, 1.5M NaCl, pH 7.5.
Scott,
We routinely make up a 10x stock of this buffer. This is our recipe:
To make 1 litre:
Maleic acid: 116.1g
NaCl: 87.66g
Water to about 800ml
At this point the maleic acid is completely insoluble. Start adding NaOH pellets while stirring the solution. Have patience, the ppt=
will not solubilize until the pH reaches about 5.5 to 6.0. At this point, start adding the pellets VERY carefully as it is easy for=
the pH to overshoot. When the pH reaches 7.5, top up with water to the correct volume and then autoclave if desired. This works eve=
ry time.
Good Luck,
Bernard Murray
recipes. However, I have to ask why this particular buffer is
recommended for this particular system? I've bought the DIG components
that I needed separately (labelling kit, antibody, substrate) and
so have never used the maleate buffer. Instead I've been using Tris-
buffered saline throughout (essentially as for Western blots with
alkaline phosphatase labelled second antibodies) and the results
seem just fine. When lazy [;-)] I've also used PBS for washing and
as long as I perform the final washes in phosphate-free buffer to
stop inhibition of the AP this works as well.
Has anyone done a comparison of maleate with anything else?
Maybe they intend this for Northern analysis and I suppose that
unlike Tris you can treat maleate with DEPC.
From a recent B-M presentation they say that the key
requirements for nice results are; 1) don't use too much probe,
and b) use a suitable membrane (they recommend their own).
As an asides, I've also heard rumours that the MgCl2 should be
ommitted from the detection buffer but this doesn't really make
sense to me. Anyone care to comment?
[No affiliation with B-M except as a customer]
Bernard Murray, Ph.D.
ber...@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Tracy Aquilla
(Bernard Murray) wrote:
>As a fellow DIG user thank you to all those that have posted working
>recipes. However, I have to ask why this particular buffer is
>recommended for this particular system? I've bought the DIG components
>that I needed separately (labelling kit, antibody, substrate) and
>so have never used the maleate buffer. Instead I've been using Tris-
>buffered saline throughout (essentially as for Western blots with
>alkaline phosphatase labelled second antibodies) and the results
>seem just fine. When lazy [;-)] I've also used PBS for washing and
>as long as I perform the final washes in phosphate-free buffer to
>stop inhibition of the AP this works as well.
> Has anyone done a comparison of maleate with anything else?
>Maybe they intend this for Northern analysis and I suppose that
>unlike Tris you can treat maleate with DEPC.
I think you got it here.
Tracy
Shoji Hatano
>..... So the tech services guys
> said to try a 5x solution. Not really a reasonable explanation, but I
> tried, and even 500mM Maleic acid precipitates at pH 2.0.
Dear Scott,
You can get the 5x solution dissolved at pH 7 or over. So you must
add NaOH until the precipitates disappear. I found 5x solution should
be adjusted to pH around 7.2. When diluted to 1x solution, we can
get the 1x solution with pH 7.5.
S. Hatano