Re: [ccp4bb] Copper Staining
One other staining method deserves to be mentioned with the others already in this thread: We use standard issue Coomassie Blue staining, but do the staining and destaining with heated solutions (place the gel in a tray on a hotplate set to ~90drg in a fumehood). We routinely have the result after 10-15 minutes (5 min stain, 5-10 min destain). -bjørn -- Bjørn Pañella Pedersen Ph.D Student, MSc Department of Molecular Biology Office: +45 89425021 University of Aarhus Lab: +45 89425010 Gustav Wieds Vej 10c Email: [EMAIL PROTECTED] DK-8000 Aarhus C Lab WWW: http://www.bioxray.dk Denmark Jacob Keller wrote: Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
You can also do this more quickly by microwaving your gels in stain/destain solutions just for the lazy or impatient Regards Mike On Wed, 2008-03-05 at 12:09 +0100, Bjørn Pañella Pedersen wrote: One other staining method deserves to be mentioned with the others already in this thread: We use standard issue Coomassie Blue staining, but do the staining and destaining with heated solutions (place the gel in a tray on a hotplate set to ~90drg in a fumehood). We routinely have the result after 10-15 minutes (5 min stain, 5-10 min destain). -bjørn DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV
[ccp4bb] Copper Staining
Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
One down side is that copper sulfate is toxic to fish and plants. Please be good to the environment and do not pour copper sulfate solutions down the drain. We normally precipitate with NaOH, filter, and put the precipitated copper in an appropriate waste. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, March 04, 2008 12:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copper Staining Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Artem P.S. I am not affiliated with Novexin in any way shape or form, but their gel stain does kick butt. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Monday, March 03, 2008 6:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copper Staining Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
Touche' Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Artem Evdokimov [EMAIL PROTECTED] To: 'Jacob Keller' [EMAIL PROTECTED]; CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 03, 2008 6:31 PM Subject: RE: [ccp4bb] Copper Staining There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Artem P.S. I am not affiliated with Novexin in any way shape or form, but their gel stain does kick butt. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Monday, March 03, 2008 6:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copper Staining Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Sounds like a good old Bradford reagent AKA colloidal Coomassie G-250. 60 mg G-250 dissolved in 50 ml ethanol and diluted into 1 L of 1/10 dilution of concentrated phosphoric acid (~8.5% final). Solubilizing agents is a smoke screen, for G-250 is fully soluble in ethanol and for the colloid particles to not penetrate the gel pores inclusion of any solubilizing agents will only be detrimental. Betcha it's a browish solution that forms blue pellet when centrifuged hard. If so, the claimed 5-10 ng sensitivity can only be achieved after 1) at least 15 min staining (30 min better; no one abolished laws of physics that requre diffusion into the gel for the interaction to take place), 2) some destaining to get rid of slight brown-blue background. Dima
