Dear Min,
regarding #1 some things come to my mind:
- What is the Kd that you got from the Biacore? And did you make sure that
the sample is concentrated enough (both on gel filtration and in
crystallization) to have a sufficient amount in the complex.
- did you use the same buffer systems?
Dear All,
Can anyone suggest me a protocol for silver-staining the PAGE that is
already stained with Coomassie.
Kris
Dear Min,
Regarding the stoichiometry that you should use in crystallizing two
proteins
that form a complex. I have looked at this question before. See:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,
Silverman, G.J., Charbonnier, J.-B. (2001)
Crystallization of
Jacob,
One of the problems with glutaraldehyde is the its chemistry is so
bizarre. It actually forms quite long transient polymers in
solution. You also have to ask yourself why formaldehyde also fixes
tissues. This is why glutaraldehyde works so well for tissue fixation
for EM as
I use my MBP with external screen, keyboard mouse all the time. The new ones
are fast, mine should easily cope with Lion
My question about Lion was because
1. on the one hand as far as I can see Bill Scott only builds latest
stand-alone Coots (0.7...) for Lion, and these don't work on 10.6
Dear Phil (and everyone):
1. I've now got automated build systems for coot for 10.7.1 and 10.6.8. I
just haven't had a chance to get the 10.6.8 one on line. I'll try to do this
today. (Also, the last few haven't build due to a change in the code with
which the compiler can't cope, but
On Thu, 2011-09-15 at 22:20 -0400, m zhang wrote:
Second is about reusing of Ni-NTA resin. According to Qiagen's
instruction, after using fresh Ni-NTA resin, one only needs to wash
the used Ni resin first with 0.5M NaOH, then with your own buffer.
After that the resin is ready to be reused
On Fri, 2011-09-16 at 15:28 +0530, K Singh wrote:
Dear All,
Can anyone suggest me a protocol for silver-staining the PAGE that is
already stained with Coomassie.
Kris
Just destain it and then use the standard silver-staining protocol. If
for some reason you want to have both stainings
Hi,
I was using Refmac from CCP4 to refine a protein's crystal structure. The
methionine has half selenium and half sulfur. I was trying to make alternate
conformations and let refmac do the refinement. But it keeps giving me error
message as follows:
There is an error in the input coordinate
Hi all,
I was using Refmac from CCP4 to refine a protein's crystal structure. The
methionine has half selenium and half sulfur. I was trying to make alternate
conformations and let refmac do the refinement. But it keeps giving me error
message as follows:
There is an error in the input
Destain it really well. One easy peasy option is to put the gel in water and
add 1 or 2 ml of cheap Q sepharose to it then boil. Gel will destain in 2-3
minutes of boiling.
On Sep 16, 2011 5:10 AM, K Singh ksc...@gmail.com wrote:
Dear All,
Can anyone suggest me a protocol for silver-staining the
1 imidazole affinity is not high which is why you use 200 mM or more to
elute. So it comes off by itself.
2 you can wash with low ph and then recharge this is somewhat easier on the
resin
On Sep 15, 2011 9:25 PM, m zhang mzhang...@hotmail.com wrote:
Dear all,
I have two questions:
First, I
Now that's a nifty trick I hadn't heard of before!
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Artem Evdokimov
Sent: Friday, September 16, 2011 10:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Silver staining Coomassie stained
It is nice indeed, that's how we stainand destain our gels in less than 8
minutes :-) there was at one time a proprietary product like tis called
magic beads or somesuch. But regular resin works ;-)
On Sep 16, 2011 9:31 AM, Soisson, Stephen M stephen_sois...@merck.com
wrote:
Now that's a nifty
On Fri, 2011-09-16 at 10:08 -0400, Ming wrote:
The methionine has half selenium and half sulfur.
Do you have the evidence that your incorporation ration was 50%?
On a practical side, try giving the SeMET a different chain ID. You can
change it back manually after refinement. Assuming that the
I did not make it clear. The protocol we used for protein expression always
limit the occ of MSE as 0.75. You may estimate your occ of MSE by ED. You
can check PDB for JCSG structures.
Cheers,
Kevin
On Fri, Sep 16, 2011 at 7:09 AM, Ming dongm...@udel.edu wrote:
Hi all,
I was using Refmac
I have a question about the bond angle restraints in the DNA cif files. I
recently submitted a protein-DNA complex to the PDB, and found out that many of
the glycosidic bond angles (atoms O4'-C1'-N9/N1) were outside the accepted
range. I switched from CCP4 6.1.13 to 6.2.0 (installed/updated
Can anyone suggest me a protocol for silver-staining the PAGE that is
already stained with Coomassie.
There is absolutely nothing wrong with silver staining of a
Coomassie-stained gel without destaining. It only prevents blowout of
most intense bands already well-visible with Coomassie. The
Hi, it depends what you mean by 'different'. The SUs of the new set are
much lower than the old values (0.8 deg vs. 3.0) so one would assume that
these are improved estimates based on new data. The difference between the
old and the new is (108.4 - 107.8) = 0.6 deg with a SU of the difference of
Hi All
I have a ligand that is dissolved in 100% DMSO. The ligand crashes out even
when diluted to 20% final DMSO concentration in the protein buffer. I have
heard of people using detergents such as n octyl Beta D glucoside to keep the
ligand solubilized at lower DMSO concentrations. Can
Hi Ritcha,
Did you try Methanol or Ethanol to see if your ligand dissolve in these
solvents? You should try these solvents first before adding any
detergents.
Good luck.
Smita Mohanty
Chaudhary, Ritcha chaudha...@missouri.edu 9/16/2011 11:04 AM
Hi All
I have a ligand that is dissolved in
To all and Alex,
-The Kd is around 500nM from Biacore.
-One of my protein tends to precipitate and is only stable below 3mg/m. If I
concentrated it to higher concentration, it will precipitate a lot after 1hr.
And it was previously crystallized at low concentration. But when I inject on
gel
Keep in mind it may also be possible to soak in
ligand from the solid if the binding constant is tight enough. Le
Chatelier's principle will drag it from the undissolved solid into
the complex. This process will be improved if you can increase
solubility in
Would be curious to know the current limitations on UV microscopy employed
for screening protein crystals - such as content of aromatic amino acids,
protein size etc.
Cheers,
Shiva
On Fri, Sep 16, 2011 at 1:19 AM, Klaus Fütterer k.futte...@bham.ac.ukwrote:
From the experience when our
Dear BB,
I hope you can help. I have been trying to density modify my potential SAD
solutions but I think it would help if I could conceptualize the arrangement of
molecules in my crystals and it is making my head hurt.
I have 2 crystal forms. I only obtained a single crystal for the first
Dear All:
I would like to know the literature on the crystal structure of
Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18).
I have the structure of I domain but not for the entire molecule. I
would greatly appreciate if people can point me to the right
article/reference.
Thanks
Hi,
On Thu, Sep 15, 2011 at 1:55 PM, Hena Dutta hdutt...@gmail.com wrote:
Hi Sabuj,
Thanks for all your answers. I finally came to this plan. Please tell me if
I am doing anything wrong.
Option 1.
HP Workstation Z400 FL998U8#ABA Desktop PC - Intel Xeon W3550 3.06GHz, 8GB
DDR3, 160GB 10k
On Fri, 2011-09-16 at 14:10 -0400, Narayanan Ramasubbu wrote:
Dear All:
I would like to know the literature on the crystal structure of
Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18).
I have the structure of I domain but not for the entire molecule. I
would greatly appreciate
Gamma delta resolvase catalytic domain stock solutions used to make a nice
clear jelly at 4 degrees, but it was perfectly reversible by warming the sample
to room T. In fact, one mutant crystallized in the stock tube after a few
trips in and out of the fridge. The crystals didn't diffract
I think I know another protein that does this: gelatin! (Well, not the
crystallization part...)
Jacob
On Fri, Sep 16, 2011 at 2:41 PM, Phoebe Rice pr...@uchicago.edu wrote:
Gamma delta resolvase catalytic domain stock solutions used to make a nice
clear jelly at 4 degrees, but it was
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