Author Topic: colloidal comassie staining  (Read 41795 times)

Luke72

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colloidal comassie staining
« on: July 14, 2004, 05:00:00 PM »
I'm looking for a protocol about colloidal comassie, can anybody help me?
thanks

Sjouke Hoving

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Re: colloidal comassie staining (Luke72)
« Reply #1 on: July 14, 2004, 05:00:00 PM »
Here we go:

According to Leigh Anderson (LSB)

* fix the gel at least 3 hrs in 50% ethanol / 3% phosphoric acid
* wash 3x20 min. in water
* pre-incubate for 1 hr in 34% methanol / 3% phosphoric acid / 17%(w/v) ammoniumsulfate solution
* add 0.35 gr Coomassie Blue (G-250) per 1 L solution and stain fo 4-5 days (after 1 day you see spots, but end-point is reached after 4-5 days, we usually stain over the weekend).
* wash the gels a few times in water to remove background stain an scan the gel

In our hands this protocol works best, better then ready-made solutions or the recently published Blue-silver from P.G. Righetti. On 1.5 mm gels detection limit of about 15 ng per spot is reached.

Sjouke


luke72

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Re: colloidal comassie staining (sjouke)
« Reply #2 on: July 14, 2004, 05:00:00 PM »
thank you very much
I try it tomorrow

Sjouke Hoving

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Re: colloidal comassie staining (luke72)
« Reply #3 on: July 14, 2004, 05:00:00 PM »
By the way,
you can put up to 5 gels in a single box (1L of solution).
Please, post your experience with the staining.

Good luck,
Sjouke


Daniel A

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Re: colloidal comassie staining (sjouke)
« Reply #4 on: July 15, 2004, 05:00:00 PM »
Dear Sjouke

regarding your Colloidal Coomassie staining protocol, you preincubate in ammonium sulfate containing solution. When you add the coomassie dye to the preincubation solution how do you do that. Do you remove the solution with a water jet vacuum pump connected to a glass flask. And then you add the coomassie to the solution and readd to the five gels. Or do you add directly the coomassie to the five gels in the box containing the preincubation solution. Do you touch the gels at all ?

best regards

Daniel A


Sjouke Hoving

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Re: colloidal comassie staining (Daniel A)
« Reply #5 on: July 18, 2004, 05:00:00 PM »
Fixing and washing solutions are easily taken out with a water-vacuum system. Then, indeed you add solid Coomassie Blue (G-250) to the ammoniumsulfate/methanol mixture. It will disperse throughout the solution when put on a rotary shaker. After staining I usually take the gels out of the box in a new box filled with distilled water (for several washes) to remove residual CCB and reduce background.
Sjouke

Vinzenz

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alternative colloidal comassie staining protokoll
« Reply #6 on: July 20, 2004, 05:00:00 PM »
I am using the following protocol which uses Aluminium Sulfate instead of Ammonium Sulfate. I compared it to a few other protocolls (including Pierce Gelcode) and found it works better. Backround is higher but I found that I can recognize spots of the same amount of protein better. I found no negativ effect on MS.
Comments on this "strange" protocoll are appreciated.
 Vinzenz

The reference is:
"Highly Sensitive and Fast Protein Detection with Coomassie Brilliant Blue in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis", Bull. Korean Chem. Soc. 2002, Vol. 23, No.11

Fix in 50% Ethanol (technical), 8% Phosphoric Acid (85%) for 3 h

Stain for at least 2 h in   200 ml         1l
0.02%  CoomassieBB G-250 (5% stock)   0.8 ml   4 ml
2% Phosphoric Acid (85 %)   4.7 ml   23.5 ml
5 % Aluminium Sulfate   10 g   50 g
10 % Ethanol (add slowly)   20 ml   100 ml

wash in water

+ quick staining
+ stable staining in water for many days (weeks)
+ sensitivity 7 ng of 40kD (or 5 ng of 66kD) protein in minigel (16 comb) easily recognized  - down to 2 ng visible
- relatively high blue backround of gels (can be reduced by washing with 10% MeOH)


LJYAN

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Re: colloidal comassie staining (sjouke)
« Reply #7 on: August 02, 2004, 05:00:00 PM »
Sjoke,

Have you tried your method on native gels? I tried the silver blue method, but it never worked. I followed the Righetti method closely but was not successful. Have no idea of what went abnormal in my hand.

Thanks,

LJYAN


Sjouke Hoving

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Re: colloidal comassie staining (LJYAN)
« Reply #8 on: August 03, 2004, 05:00:00 PM »
No, but I use our method on 1-D and 2-D gels. You can even stain with silver afterwards. I was also not succesful with the Righetti protocol (Blue silver). In their paper they compare however with the original Neuhoff protocol and not with the more sensitive Anderson method that we use (see protocol in post above).
Sjouke

susann

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Re: colloidal comassie staining (sjouke)
« Reply #9 on: August 10, 2004, 05:00:00 PM »
Hi,
I have been using Pierce's Gelcode Blue stain on the recommendation of our campus protein/ms facility.  Does anyone have any thoughts on how good gelcode blue performs ...should I consider a change to the staining protocol above (the first one) and is that protocol usable in MS analysis?  Some of my gels are posted under the expired strips and IPG buffer heading in the discussion.
Thanks!
Susanne

Sjouke Hoving

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Re: colloidal comassie staining (susann)
« Reply #10 on: August 11, 2004, 05:00:00 PM »
If you want to save money and have a good staining, make your own colloidal stain. It is cheap, it is MS compatible, it is sensitive... what more would you want???

Sjouke


wenlei

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Re: colloidal comassie staining (sjouke)
« Reply #11 on: August 18, 2004, 05:00:00 PM »
Hi-, sjouke,

Thank you very much for your reciept.
I have try once. But I fould it is very hard to dissolve 17% ammonium sulfate in the solution.
How do you solve this problem.

Best regards,

Wenlei


LRC

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Re: colloidal comassie staining (wenlei)
« Reply #12 on: August 18, 2004, 05:00:00 PM »
I am trying this method (methonal/ammonium sulfate)today, I also found that 170 g ammonium sulfate is too much for 1L solution and CBB g250 does't disolve well neither. However, the staining is very good.

LRC


Sjouke Hoving

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Re: colloidal comassie staining
« Reply #13 on: August 19, 2004, 05:00:00 PM »
First you dissolve the ammoniumsulfate in water/phosphoric acid and then you add slowly the methanol. This works fine.

It's a colloidal solution - the Coomassie is not supposed to dissolve, but should be dispersed throughout the solution  

Sjouke


Peter Campbell

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Re: colloidal comassie staining (sjouke)
« Reply #14 on: August 22, 2004, 05:00:00 PM »
When I try making colloidal Coomassie stain with the dye added to the acid/ammonium sulphate solution before the methanol is added the final solution has coarse grains of dye that quickly fall to the bottom of the container.  I understand that "colloidal" doesn't mean "in solution" but I thought the word meant really fine particles that don't settle so quickly.  I can get something that works as a stain if I dissolve the coomassie in the methanol and then add that solution to the acid/ammonium sulphate/water solution but this is contrary to all version of the recipe.  My version looks blue and the dye doesn't settle whereas the standard version, in my hands, looks brown and gritty.  Is that really what a proper colloidal stain looks like or am I not getting what others get when adding things in the conventional order?  
Peter.