Re: [ccp4bb] Pilatus Issues

[Keller, Jacob]

Regarding daftness, it seems that the detector is wider than tall, which should 
improve the ratio of Lorentz-problematic reflections to good, fast-moving ones. 
So I assume it was a choice between that and excluding some spots in the 
gap--an appropriate calculation could be done to see whether this is 
appropriate.

I was curious about the polarization issue you mentioned: how does horizontal 
orientation speak to polarization? I don't understand the connection.

All the best,

Jacob Keller



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerard 
Bricogne
Sent: Saturday, July 15, 2017 5:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Pilatus Issues

Dear John,

 Having just seen Andreas's message regarding the best source of support to 
address your enquiry, I have a further remark to make about your instrument.

 As this is a lab instrument, the Omega axis would be vertical, and indeed 
the beam stop shadow (vertical on the top module) and the diffuse shadow of the 
sample holder (vertical on the bottom module) would confirm this. This being 
the case, it is quite simply *daft* to have the gap between the two modules 
being horizontal. That is done on purpose on synchrotron beamlines because of 
the polarisation of the beam (which is why Omega is horizontal on such 
beamlines), but in a lab system the gap should be in the vertical direction. As 
currently placed in your system, this gap is cutting into perfectly good data, 
whereas if it were vertical instead, it would only cut out data that are 
getting perilouly close to the cusp anyway.

 You should ask the manufacturer of your diffractometer to rotate your 
detector by 90 degrees! Someone in the OEM world forgot about the Lorentz 
factor ;-) .


 With best wishes,
 
  Gerard.

--
On Fri, Jul 14, 2017 at 05:14:03PM +0100, John Hardin wrote:
> Hi,
> 
> We have recently noticed an issue with our Pilatus (biased pixels/vertical 
> lines).
> I was curious as to whether anyone else has seen this or might know what 
> could have caused it?
> 
> Best,
> John
> 

Re: [ccp4bb] Pilatus Issues

[John Hardin]

Thank you Gerard.

This seems to be a very valid point. 
I myself cannot fully rule out the possibility that greater minds than mine 
may present compelling arguments for installation with a horizontal gap 
orientation.
Yet… taking your point, it is admittedly difficult for me to see any advantage 
to the current orientation other than ease of installation (mounting bracket 
location).

Thank you very much for your advice.

Best,
John

Re: [ccp4bb] Pilatus Issues

[John Hardin]

Thank you for the advice.

I had hoped to ascertain the frequency of this issue amongst the user base
and perhaps learn whether there is anything at all that we users can do to 
reduce the likelihood of its reoccurrence.

I suspect that this is a rare issue and that it is likely an electronics 
problem 
and as such is probably outside of user control/influence (aside from 
maintaining proper temperature and humidity control) .

Best,
John

Re: [ccp4bb] Pilatus Issues

[Gerard Bricogne]

Dear John,

 Having just seen Andreas's message regarding the best source of
support to address your enquiry, I have a further remark to make about
your instrument.

 As this is a lab instrument, the Omega axis would be vertical,
and indeed the beam stop shadow (vertical on the top module) and the
diffuse shadow of the sample holder (vertical on the bottom module)
would confirm this. This being the case, it is quite simply *daft* to
have the gap between the two modules being horizontal. That is done on
purpose on synchrotron beamlines because of the polarisation of the
beam (which is why Omega is horizontal on such beamlines), but in a
lab system the gap should be in the vertical direction. As currently
placed in your system, this gap is cutting into perfectly good data,
whereas if it were vertical instead, it would only cut out data that
are getting perilouly close to the cusp anyway.

 You should ask the manufacturer of your diffractometer to rotate
your detector by 90 degrees! Someone in the OEM world forgot about the
Lorentz factor ;-) .


 With best wishes,
 
  Gerard.

--
On Fri, Jul 14, 2017 at 05:14:03PM +0100, John Hardin wrote:
> Hi,
> 
> We have recently noticed an issue with our Pilatus (biased pixels/vertical 
> lines).
> I was curious as to whether anyone else has seen this or might know what 
> could have caused it?
> 
> Best,
> John
> 

Re: [ccp4bb] Pilatus Issues

[Andreas Förster]

Dear John,

I can't answer your questions with the limited information you supply, but
I would recommend contacting the company that sold you the detector.  Most
probably that's the maker of your diffractometer.  They'll be able to help
you out.  If it's something that requires repair, they might even send you
a loan instrument while taking care of yours.  If you bought the detector
directly from Dectris, please send an email to our support team with
details.

All best.


Andreas



On Fri, Jul 14, 2017 at 6:14 PM, John Hardin  wrote:

> Hi,
>
> We have recently noticed an issue with our Pilatus (biased pixels/vertical
> lines).
> I was curious as to whether anyone else has seen this or might know what
> could have caused it?
>
> Best,
> John
>
>
>


-- 

Andreas Förster, Ph.D.
MX Application Scientist, Scientific Sales
Phone: +41 56 500 2100 | Direct: +41 56 500 21 76 | Email:
andreas.foers...@dectris.com
DECTRIS Ltd. | Taefernweg 1 | 5405 Baden-Daettwil | Switzerland |
www.dectris.com

[image: LinkedIn] 
 [image: facebook]







Confidentiality Note: This message is intended only for the use of the
named
recipient(s) and may contain confidential and/or privileged information. If
you
are not the intended recipient, please contact the sender and delete the
message.
Any unauthorized use of the information contained in this message is
prohibited.

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Nicholas Larsen]

Did anyone suggest adding any known ligand?  If your protein happens to
bind some compound/peptide/DNA/RNA/whatever, including that other partner
could dramatically change it's solution properties and be an easy fix for
your handling/formulation issues.
Good luck!

On Fri, Jul 14, 2017 at 6:33 AM, Chris Fage  wrote:

> Dear All,
>
> Thank you for the many suggestions. After sending my first message to the
> BB, I tried exchanging the sample into buffer containing 5 mM EDTA and also
> into buffer at pH 9.0 (using BICINE). Neither of these appear to have
> helped the instability--precipitation still occurred within ~1 min of
> removal of the tube from ice. The theoretical pI is ~6.1, which is far from
> my working pH, although as Mark indicated the calculated pI may be
> inaccurate, and I may even need to try a more acidic pH. I will test the
> ideas provided by everyone over the next week and leave some feedback. It
> may be that I need to prepare a different truncation, as this domain is
> excised from a larger covalent assembly. I have purified homologs trimmed
> at a similar position in the past, but of course that doesn't guarantee
> good behavior in my current system.
>
> Best,
> Chris
>
> On Fri, Jul 14, 2017 at 10:20 AM, Eugene Osipov 
> wrote:
>
>>  Hi, try to add 1-5 mM EDTA, because trace amounts of metals cause
>> precipitation of tagged proteins.
>>
>> 14 июля 2017 г. 1:40 пользователь "Chris Fage" 
>> написал:
>>
>> Dear CCP4BB Community,
>>>
>>> This week, I purified a nicely overexpressing protein by Ni-NTA followed
>>> by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
>>> fractions to ~1 mL, transferred the spin filter to ice, and then collected
>>> 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
>>> heavily in the pipet tip before I could dispense it onto the Nanodrop
>>> pedestal, directly adjacent to my ice box. This effect seems to be abated
>>> at 4 C, as the protein remained stable in cold room-chilled pipet tips.
>>> However, the protein also precipitated heavily when overnight at 4 C in 1
>>> mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
>>> overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
>>> 10% glycerol) prior to gel filtration. Has anyone experienced and resolved
>>> a similar issue before? Do any useful additives come to mind?
>>>
>>> Things I have tried with the gel filtration sample:
>>> -Exchanging buffer to restore the salt concentration to Ni-NTA levels
>>> (e.g. 500 mM).
>>> -Exchanging buffer to add 10% glycerol.
>>> -Simply diluting the protein in gel filtration buffer to rule out
>>> concentration dependence.
>>>
>>> In each case, the protein precipitates to a milky solution within about
>>> a minute of removal from ice (I am working with 20-50 uL volumes in PCR
>>> tubes).
>>>
>>> Many thanks for any suggestions!
>>>
>>> Best,
>>> Chris
>>>
>>
>

-- 
[This e-mail message may contain privileged, confidential and/or 
proprietary information of H3 Biomedicine. If you believe that it has been 
sent to you in error, please contact the sender immediately and delete the 
message including any attachments, without copying, using, or distributing 
any of the information contained therein. This e-mail message should not be 
interpreted to include a digital or electronic signature that can be used 
to authenticate an agreement, contract or other legal document, nor to 
reflect an intention to be bound to any legally-binding agreement or 
contract.]

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Kevin Jin]

Hi Chris,

In your Ni-NTA buffer, the total [Na+] could be greater than 530mM after
titration. Likely, your protein likes higher concentration salt for surface
charge stabilization. Adding Glycerol may further modify the charge
distribution and make the protein happy in the solution. For further
verification, I usually try the buffer without Glycerin or replace it with
small PEG (PEG 100) for +/- impacts, respectively. Indeed, it is pretty
good starting point for crystallization.

I noticed that HEPES is also slightly temperature dependent. In most of the
cases, the gap could be ignored. It may be helpful. In my previous cases, I
tried PBS and Bis-tris with the same pH. The stability was improved great.

Can you keep every thing same but different kind of buffer?

Hope it would be helpful.

Sincerely.



On Thu, Jul 13, 2017 at 3:40 PM, Chris Fage  wrote:

> Dear CCP4BB Community,
>
> This week, I purified a nicely overexpressing protein by Ni-NTA followed
> by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
> fractions to ~1 mL, transferred the spin filter to ice, and then collected
> 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
> heavily in the pipet tip before I could dispense it onto the Nanodrop
> pedestal, directly adjacent to my ice box. This effect seems to be abated
> at 4 C, as the protein remained stable in cold room-chilled pipet tips.
> However, the protein also precipitated heavily when overnight at 4 C in 1
> mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
> overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
> 10% glycerol) prior to gel filtration. Has anyone experienced and resolved
> a similar issue before? Do any useful additives come to mind?
>
> Things I have tried with the gel filtration sample:
> -Exchanging buffer to restore the salt concentration to Ni-NTA levels
> (e.g. 500 mM).
> -Exchanging buffer to add 10% glycerol.
> -Simply diluting the protein in gel filtration buffer to rule out
> concentration dependence.
>
> In each case, the protein precipitates to a milky solution within about a
> minute of removal from ice (I am working with 20-50 uL volumes in PCR
> tubes).
>
> Many thanks for any suggestions!
>
> Best,
> Chris
>



-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/