Re: [ccp4bb] KCl in SDS-PAGE workarounds?

2019-03-03 Thread Artem Evdokimov 
K+ and NH4+ ions precipitate SDS - forming the lovely cheese that makes it
very difficult to load and run those samples.

Sometimes using LDS can help with the potassium issue. Heating the sample
right before loading might help as well, or using a lot larger ratio of
buffer to sample.

Precipitation of protein prior to the gel is certainly an option, although
some proteins have difficultues with recovering after that (membrane
proteins in particular).

Artem

- Cosmic Cats approve of this message


On Sat, Mar 2, 2019 at 9:53 PM Keller, Jacob 
wrote:

> Dear crystallographers,
>
>
>
> It has been my experience that KCl does nasty things when loading SDS-PAGE
> gels. Does anyone have an easy workaround, perhaps TCA precipitation?
> Ideally this would be something nicely quantitative yet quick and easy….
>
>
>
> Any suggestions appreciated.
>
>
>
> All the best,
>
>
>
> Jacob Keller
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
>
>
> *From:* CCP4 bulletin board  * On Behalf Of *Artem
> Evdokimov
> *Sent:* Saturday, March 2, 2019 8:32 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] ionic strength for extraction buffer of membrane
> proteins
>
>
>
> Hi Alex,
>
>
>
> In my experience you just have to experiment with these parameters (in
> however of a limited space that is afforded by your experimental set-up).
> If you have the right tools you can slog through semifactorial or DoE-based
> scouting of various parameters with relative ease. These parameters
> typically include pH, salinity, detergent(s), and other variables (e.g. the
> nature of the buffer and salt(s) - many cases exist where the 'standard'
> choices do not work well).
>
>
>
> Literature-based analogies do help on occasion. For example,
> membrane-spanning p450-type proteins often prefer high salt, and often
> would do better in sodium acetate as opposed to chloride. I've worked with
> ion channels that preferred 3M NaCl, or 2M MgCl2 and other fairly weird
> conditions...
>
>
>
> Artem
>
> - Cosmic Cats approve of this message
>
>
>
>
>
> On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín <
> aperalva...@gmail.com> wrote:
>
> Dear all,
>
> any reference as a guide for selecting the appropriate salt and
> concentration for membrane proteins extraction buffer?
>
> Best,
>
> Alex
>
> --
> Alex Perálvarez-Marín, Ph.D.
> Centre d'Estudis en Biofísica / Unitat de Biofísica
> Edifici M
> Universitat Autònoma de Barcelona
> 08193 Cerdanyola del Vallés
> Barcelona
> Spain
> Phone: +34 93 581 4504
> FAX:  +34 93 581 1907
> e-mail: aperalva...@gmail.com
> LinkedIn: es.linkedin.com/in/aperalvarez/
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] KCl in SDS-PAGE workarounds?

2019-03-03 Thread stephen.c...@rc-harwell.ac.uk 
Hi Jacob,

If you keep the protein solution warm the KDS  remains soluble. Just load the 
sample on the gel immediately after heating to denature.  This has allowed me 
to load samples containing up to 1.5M KCl with no problems.

Best wishes,

Steve

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

From: CCP4 bulletin board  on behalf of Bonsor, Daniel 

Sent: 03 March 2019 03:19:34
To: ccp4bb
Subject: Re: [ccp4bb] KCl in SDS-PAGE workarounds?


SDS is soluble, whereas the potassium salt of dodecyl sulphate is insoluble. 
You could try 18-Crown-6 ether to chelate the potassium, though I don't know if 
the crown ether would then affect the gel.


Dan


Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201



From: CCP4 bulletin board  on behalf of Keller, Jacob 

Sent: Saturday, March 2, 2019 9:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] KCl in SDS-PAGE workarounds?


Dear crystallographers,



It has been my experience that KCl does nasty things when loading SDS-PAGE 
gels. Does anyone have an easy workaround, perhaps TCA precipitation? Ideally 
this would be something nicely quantitative yet quick and easy….



Any suggestions appreciated.



All the best,



Jacob Keller



+

Jacob Pearson Keller

Research Scientist / Looger Lab

HHMI Janelia Research Campus

19700 Helix Dr, Ashburn, VA 20147

Desk: (571)209-4000 x3159

Cell: (301)592-7004

+



The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.



From: CCP4 bulletin board  On Behalf Of Artem Evdokimov
Sent: Saturday, March 2, 2019 8:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins



Hi Alex,



In my experience you just have to experiment with these parameters (in however 
of a limited space that is afforded by your experimental set-up). If you have 
the right tools you can slog through semifactorial or DoE-based scouting of 
various parameters with relative ease. These parameters typically include pH, 
salinity, detergent(s), and other variables (e.g. the nature of the buffer and 
salt(s) - many cases exist where the 'standard' choices do not work well).



Literature-based analogies do help on occasion. For example, membrane-spanning 
p450-type proteins often prefer high salt, and often would do better in sodium 
acetate as opposed to chloride. I've worked with ion channels that preferred 3M 
NaCl, or 2M MgCl2 and other fairly weird conditions...



Artem

- Cosmic Cats approve of this message





On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín 
mailto:aperalva...@gmail.com>> wrote:

Dear all,

any reference as a guide for selecting the appropriate salt and
concentration for membrane proteins extraction buffer?

Best,

Alex

--
Alex Perálvarez-Marín, Ph.D.
Centre d'Estudis en Biofísica / Unitat de Biofísica
Edifici M
Universitat Autònoma de Barcelona
08193 Cerdanyola del Vallés
Barcelona
Spain
Phone: +34 93 581 4504
FAX:  +34 93 581 1907
e-mail: aperalva...@gmail.com<mailto:aperalva...@gmail.com>
LinkedIn: 
es.linkedin.com/in/aperalvarez/<http://es.linkedin.com/in/aperalvarez/>



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Re: [ccp4bb] KCl in SDS-PAGE workarounds?

2019-03-02 Thread Bonsor, Daniel 
SDS is soluble, whereas the potassium salt of dodecyl sulphate is insoluble. 
You could try 18-Crown-6 ether to chelate the potassium, though I don't know if 
the crown ether would then affect the gel.


Dan


Daniel A. Bonsor PhD
Institute of Human Virology,
University of Maryland at Baltimore
725 W. Lombard Street N571
Baltimore
MD 21201



From: CCP4 bulletin board  on behalf of Keller, Jacob 

Sent: Saturday, March 2, 2019 9:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] KCl in SDS-PAGE workarounds?


Dear crystallographers,



It has been my experience that KCl does nasty things when loading SDS-PAGE 
gels. Does anyone have an easy workaround, perhaps TCA precipitation? Ideally 
this would be something nicely quantitative yet quick and easy….



Any suggestions appreciated.



All the best,



Jacob Keller



+

Jacob Pearson Keller

Research Scientist / Looger Lab

HHMI Janelia Research Campus

19700 Helix Dr, Ashburn, VA 20147

Desk: (571)209-4000 x3159

Cell: (301)592-7004

+



The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.



From: CCP4 bulletin board  On Behalf Of Artem Evdokimov
Sent: Saturday, March 2, 2019 8:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins



Hi Alex,



In my experience you just have to experiment with these parameters (in however 
of a limited space that is afforded by your experimental set-up). If you have 
the right tools you can slog through semifactorial or DoE-based scouting of 
various parameters with relative ease. These parameters typically include pH, 
salinity, detergent(s), and other variables (e.g. the nature of the buffer and 
salt(s) - many cases exist where the 'standard' choices do not work well).



Literature-based analogies do help on occasion. For example, membrane-spanning 
p450-type proteins often prefer high salt, and often would do better in sodium 
acetate as opposed to chloride. I've worked with ion channels that preferred 3M 
NaCl, or 2M MgCl2 and other fairly weird conditions...



Artem

- Cosmic Cats approve of this message





On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín 
mailto:aperalva...@gmail.com>> wrote:

Dear all,

any reference as a guide for selecting the appropriate salt and
concentration for membrane proteins extraction buffer?

Best,

Alex

--
Alex Perálvarez-Marín, Ph.D.
Centre d'Estudis en Biofísica / Unitat de Biofísica
Edifici M
Universitat Autònoma de Barcelona
08193 Cerdanyola del Vallés
Barcelona
Spain
Phone: +34 93 581 4504
FAX:  +34 93 581 1907
e-mail: aperalva...@gmail.com<mailto:aperalva...@gmail.com>
LinkedIn: 
es.linkedin.com/in/aperalvarez/<http://es.linkedin.com/in/aperalvarez/>



To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] KCl in SDS-PAGE workarounds?

2019-03-02 Thread Keller, Jacob 
Dear crystallographers,

It has been my experience that KCl does nasty things when loading SDS-PAGE 
gels. Does anyone have an easy workaround, perhaps TCA precipitation? Ideally 
this would be something nicely quantitative yet quick and easy….

Any suggestions appreciated.

All the best,

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board  On Behalf Of Artem Evdokimov
Sent: Saturday, March 2, 2019 8:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins

Hi Alex,

In my experience you just have to experiment with these parameters (in however 
of a limited space that is afforded by your experimental set-up). If you have 
the right tools you can slog through semifactorial or DoE-based scouting of 
various parameters with relative ease. These parameters typically include pH, 
salinity, detergent(s), and other variables (e.g. the nature of the buffer and 
salt(s) - many cases exist where the 'standard' choices do not work well).

Literature-based analogies do help on occasion. For example, membrane-spanning 
p450-type proteins often prefer high salt, and often would do better in sodium 
acetate as opposed to chloride. I've worked with ion channels that preferred 3M 
NaCl, or 2M MgCl2 and other fairly weird conditions...

Artem
- Cosmic Cats approve of this message


On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín 
mailto:aperalva...@gmail.com>> wrote:
Dear all,

any reference as a guide for selecting the appropriate salt and
concentration for membrane proteins extraction buffer?

Best,

Alex

--
Alex Perálvarez-Marín, Ph.D.
Centre d'Estudis en Biofísica / Unitat de Biofísica
Edifici M
Universitat Autònoma de Barcelona
08193 Cerdanyola del Vallés
Barcelona
Spain
Phone: +34 93 581 4504
FAX:  +34 93 581 1907
e-mail: aperalva...@gmail.com
LinkedIn: 
es.linkedin.com/in/aperalvarez/



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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