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
>>>
>>
>

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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/

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Chun Luo]

Hi Chris,

Does you protein polymerize?

We had examples that protein solution turned turbid at high temperature and 
become aggregates. We also had proteins precipitated on ice but not at 4C. 
Sometimes not much protein loss after clearance.

Looks like your Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) 
works. It should work well with crystallization.

Cheers,

Chun 

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Fage
Sent: Friday, July 14, 2017 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein rapidly precipitates when off ice

 

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 <e.m.osi...@gmail.com 
<mailto:e.m.osi...@gmail.com> > 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" <fage...@gmail.com 
<mailto:fage...@gmail.com> > 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 

 

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Zhang, Yuzhu]

I did not follow all the responses to this, but, did you try to use the Ni-NTA 
buffer (500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) to run the gel 
filtration?

Yuzhu.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Fage
Sent: Friday, July 14, 2017 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein rapidly precipitates when off ice

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 
<e.m.osi...@gmail.com<mailto:e.m.osi...@gmail.com>> 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" 
<fage...@gmail.com<mailto:fage...@gmail.com>> 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





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Re: [ccp4bb] Protein rapidly precipitates when off ice

[Chris Fage]

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
>>
>

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Vicky Tsirkone]

Dear all,

I will give my advice once again and hopefully you will discuss on it the
next 2 days :P
Chris might be worth trying a 2ndary or 3ary structure prediction tool to
ensure that you do not have f.i. flexible ends.
Disordered regions may lead to this kind of issues, as well.
Personally I love HHPRED. Previous experience on closely homologous
proteins may give you a good idea on how to treat your protein.

Good luck!

Vicky

On Fri, Jul 14, 2017 at 9:21 AM, Mark J van Raaij <mjvanra...@cnb.csic.es>
wrote:

> I'd try varying the pH independent of the theoretical pI, sometimes the
> real pI is very different.
> (I've worked on a protein with theoretical pI 9.2, real pI determined by
> iso-electric focussing 7.8).
>
> I'd also try limited proteolysis on the milky sample and see if you can
> solubilise it while a sufficiently interesting protein fragment remains.
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
>
> On 14 Jul 2017, at 08:14, Debanu Das <debanu@gmail.com> wrote:
>
> Hi,
>
> I was in Sung-Hou Kim's group when this work below was performed and
> published and I also tried it out on many occasions. Elegant piece of
> work and certainly worth trying.
>
> Aside from the suggestions of trying different pH and related optimum
> solubility screening and if higher salt and glycerol are not helping
> when off ice, you can consider the following:
>
> a) Try not concentrating the protein and/or reducing the expression
> levels. Maybe you do not need to have so much protein if it leads to
> relatively rapid precipitation.
>
> b) Set up some crystallization screens with the protein before
> concentration, especially if the protein is clean enough after Ni-NTA.
> We crystallized many proteins with Ni-NTA followed by tag cleavage,
> and second IMAC
>
> c) Do a high speed spin of the precipitated sample to remove the
> precipitate, and run on a gel to verify sample, estimate concentration
> and set up crystallization screens on that. This is related to (a) to
> remove excess protein.
>
> d) set up crystallization screens at 4C immediately or over a few
> hours if stabilized by higher salt/glycerol and maybe the chemicals in
> the crystallization reagents can stabilize the protein.
>
> e) If it is a nucleic acid binding protein, try complexes with nucleic
> acid added during purification or right after at 4C. Or try protein
> partners or other ligands.
>
> f) is the protein Cys rich? Can you anticipate/estimate or model
> surface exposed Cys or S-S bonds? Do you have adequate reducing agent
> in the sample?
>
> g) Lastly, more esoteric stuff like construct and vector optimization,
> mutations, etc.
>
> I am sure there may be a few other things you can try and there may be
> more suggestions here.
>
> Best,
> Debanu
> --
> Debanu Das
>
> On Thu, Jul 13, 2017 at 10:46 PM, Briggs, David C
> <david.bri...@imperial.ac.uk> wrote:
>
> Hi Chris,
>
> What is the theoretical pI of your protein? If it is around pH 7.5, you
> might try gel filtering your protein into a different buffer/pH
> combination.
> Try changing by at least 1 pH unit in either direction.
>
> If the pI isn't a problem, then you might try try solubility screening as
> outlined...
>
> http://scripts.iucr.org/cgi-bin/paper?dz5020
>
> HTH,
>
> Dave
>
> --
> Dr David C Briggs
> Hohenester Lab
> Department of Life Sciences
> Imperial College London
> UK
> http://about.me/david_briggs
>
> 
> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Chris Fage
> <fage...@gmail.com>
> Sent: Thursday, July 13, 2017 11:40:34 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Protein rapidly precipitates when off ice
>
> 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 HE

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Mark J van Raaij]

I'd try varying the pH independent of the theoretical pI, sometimes the real pI 
is very different.
(I've worked on a protein with theoretical pI 9.2, real pI determined by 
iso-electric focussing 7.8).

I'd also try limited proteolysis on the milky sample and see if you can 
solubilise it while a sufficiently interesting protein fragment remains.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

> On 14 Jul 2017, at 08:14, Debanu Das <debanu@gmail.com> wrote:
> 
> Hi,
> 
> I was in Sung-Hou Kim's group when this work below was performed and
> published and I also tried it out on many occasions. Elegant piece of
> work and certainly worth trying.
> 
> Aside from the suggestions of trying different pH and related optimum
> solubility screening and if higher salt and glycerol are not helping
> when off ice, you can consider the following:
> 
> a) Try not concentrating the protein and/or reducing the expression
> levels. Maybe you do not need to have so much protein if it leads to
> relatively rapid precipitation.
> 
> b) Set up some crystallization screens with the protein before
> concentration, especially if the protein is clean enough after Ni-NTA.
> We crystallized many proteins with Ni-NTA followed by tag cleavage,
> and second IMAC
> 
> c) Do a high speed spin of the precipitated sample to remove the
> precipitate, and run on a gel to verify sample, estimate concentration
> and set up crystallization screens on that. This is related to (a) to
> remove excess protein.
> 
> d) set up crystallization screens at 4C immediately or over a few
> hours if stabilized by higher salt/glycerol and maybe the chemicals in
> the crystallization reagents can stabilize the protein.
> 
> e) If it is a nucleic acid binding protein, try complexes with nucleic
> acid added during purification or right after at 4C. Or try protein
> partners or other ligands.
> 
> f) is the protein Cys rich? Can you anticipate/estimate or model
> surface exposed Cys or S-S bonds? Do you have adequate reducing agent
> in the sample?
> 
> g) Lastly, more esoteric stuff like construct and vector optimization,
> mutations, etc.
> 
> I am sure there may be a few other things you can try and there may be
> more suggestions here.
> 
> Best,
> Debanu
> --
> Debanu Das
> 
> On Thu, Jul 13, 2017 at 10:46 PM, Briggs, David C
> <david.bri...@imperial.ac.uk> wrote:
>> Hi Chris,
>> 
>> What is the theoretical pI of your protein? If it is around pH 7.5, you
>> might try gel filtering your protein into a different buffer/pH combination.
>> Try changing by at least 1 pH unit in either direction.
>> 
>> If the pI isn't a problem, then you might try try solubility screening as
>> outlined...
>> 
>> http://scripts.iucr.org/cgi-bin/paper?dz5020
>> 
>> HTH,
>> 
>> Dave
>> 
>> --
>> Dr David C Briggs
>> Hohenester Lab
>> Department of Life Sciences
>> Imperial College London
>> UK
>> http://about.me/david_briggs
>> 
>> 
>> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Chris Fage
>> <fage...@gmail.com>
>> Sent: Thursday, July 13, 2017 11:40:34 PM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: [ccp4bb] Protein rapidly precipitates when off ice
>> 
>> 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

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Debanu Das]

Hi,

I was in Sung-Hou Kim's group when this work below was performed and
published and I also tried it out on many occasions. Elegant piece of
work and certainly worth trying.

Aside from the suggestions of trying different pH and related optimum
solubility screening and if higher salt and glycerol are not helping
when off ice, you can consider the following:

a) Try not concentrating the protein and/or reducing the expression
levels. Maybe you do not need to have so much protein if it leads to
relatively rapid precipitation.

b) Set up some crystallization screens with the protein before
concentration, especially if the protein is clean enough after Ni-NTA.
We crystallized many proteins with Ni-NTA followed by tag cleavage,
and second IMAC

c) Do a high speed spin of the precipitated sample to remove the
precipitate, and run on a gel to verify sample, estimate concentration
and set up crystallization screens on that. This is related to (a) to
remove excess protein.

d) set up crystallization screens at 4C immediately or over a few
hours if stabilized by higher salt/glycerol and maybe the chemicals in
the crystallization reagents can stabilize the protein.

e) If it is a nucleic acid binding protein, try complexes with nucleic
acid added during purification or right after at 4C. Or try protein
partners or other ligands.

f) is the protein Cys rich? Can you anticipate/estimate or model
surface exposed Cys or S-S bonds? Do you have adequate reducing agent
in the sample?

g) Lastly, more esoteric stuff like construct and vector optimization,
mutations, etc.

I am sure there may be a few other things you can try and there may be
more suggestions here.

Best,
Debanu
--
Debanu Das

On Thu, Jul 13, 2017 at 10:46 PM, Briggs, David C
<david.bri...@imperial.ac.uk> wrote:
> Hi Chris,
>
> What is the theoretical pI of your protein? If it is around pH 7.5, you
> might try gel filtering your protein into a different buffer/pH combination.
> Try changing by at least 1 pH unit in either direction.
>
> If the pI isn't a problem, then you might try try solubility screening as
> outlined...
>
> http://scripts.iucr.org/cgi-bin/paper?dz5020
>
> HTH,
>
> Dave
>
> --
> Dr David C Briggs
> Hohenester Lab
> Department of Life Sciences
> Imperial College London
> UK
> http://about.me/david_briggs
>
> 
> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Chris Fage
> <fage...@gmail.com>
> Sent: Thursday, July 13, 2017 11:40:34 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Protein rapidly precipitates when off ice
>
> 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

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Briggs, David C]

Hi Chris,

What is the theoretical pI of your protein? If it is around pH 7.5, you might 
try gel filtering your protein into a different buffer/pH combination. Try 
changing by at least 1 pH unit in either direction.

If the pI isn't a problem, then you might try try solubility screening as 
outlined...

http://scripts.iucr.org/<http://scripts.iucr.org/cgi-bin/paper?dz5020>cgi<http://scripts.iucr.org/cgi-bin/paper?dz5020>-bin/paper?dz5020<http://scripts.iucr.org/cgi-bin/paper?dz5020>

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Chris Fage 
<fage...@gmail.com>
Sent: Thursday, July 13, 2017 11:40:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein rapidly precipitates when off ice

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

[ccp4bb] Protein rapidly precipitates when off ice

[Chris Fage]

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