How do you remove Coomassie stains from membranes?
Remove stain with Eraser/Methanol solution for 10 min for PVDF and 2 min for nitrocellulose at room temperature on a rotary shaker. Certain proteins may require more time (up to 20 min) to completely remove MemCode stain. Briefly rinse membrane with water five times then proceed with immunoblotting.
How do you Destain a Coomassie?
Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear.
Can you transfer a gel after Coomassie?
Coomassie stain Incubate for 4 h to overnight at room temperature on a shaker. Transfer the gel (save the dye mixture; it can be re-used many times) to a mixture of 67.5% distilled water, 7.5% acetic acid, and 25% methanol, place on shaker, and replace with fresh rinse mixture until the excess dye has been removed.
How do you Destain Ponceau?
Remove the Ponceau S stain from the protein bands using 200 μM NaOH/20% acetonitrile for 1 minute. Wash the membrane three times with water for 5 minutes each time, then air day it.
Can you Coomassie stain a PVDF membrane?
Coomassie R-250: Stain PVDF membrane with 0.1% Coomassie R-250 in 40% MeOH for no longer than ONE MINUTE usually 15 to 20 seconds will suffice. (Staining for longer periods of time will result in high background and will interfere with extraction and cleavage)
How do you remove Coomassie gel stain?
The Coomassie stain is removed by aspiration after staining. 4. Cover the gel with ~250mL of the destain solution and allow the gel to destain with gentle agitation. The destain solution should be changed several times, removing it at each change by aspiration.
How do you prepare a Destain solution?
Destain: Add 500mL of HPLC- grade methanol to 300 mL of HPLC grade water. Add 100 mL of reagent grade acetic acid and, after mixing, adjust the total volume to 1000mL with water. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid.
Can you Destain Coomassie overnight?
Just wash the gel after the destain to remove any destain remaining and put the gel back into coommassie stain. Then destain as usual. If you destain overnight, you can also decrease the time in destain so that your bands are still dark when the background has destained sufficiently.
Can you over stain with Coomassie?
Silver staining over Coomassie-stain can work, but is usually quite ugly. If you can see your bands (even faintly) on Coomassie, I would run a separate gel with 10-30% sample and silver stain from fresh – it is about 10-100x more sensitive.
How do you make Coomassie Blue stain?
To prepare 1 l of a staining solution, dissolve 1 g of Coomassie R250 to 300 ml of methanol. Then add 650 ml of MQ water and 50 ml of acetic acid. Stir the solution on a magnetic stirrer for 2 h. The solution can be filtered through a Whatman No.
What is Coomassie blue staining?
Description. Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent (“destaining”). This treatment allows the visualization of proteins as blue bands on a clear background.
Can you Western blot a Coomassie stained gel?
The answer is yes: western blotting Coomassie-stained proteins can be done, but it’s not a simple or efficient process. As you know, there are two types of Coomassie stains – “classical” and “colloidal”. Proteins stained by one of these two methods will behave differently if you try to blot them afterwards.
How to destain SDS-PAGE gel with Coomassie stain?
Remove SDS-PAGE gel from glass and rinse once in ddH2O in a suitable container with a lid. Try not to use a container much larger or much smaller then the gel. 2) Add enough Coomassie Stain to cover the gel by 1/2 inch (~ 1.5 cm). 3) Microwave on high power for 40 seconds to 1 minute (until the Coomassie Stain boils).
How to destain glass with a Coomassie stain?
1) Remove SDS-PAGE gel from glass and rinse 2) Add enough Coomassie Stain to cover the 3) Microwave on high power for 40 seconds t 4) Incubate the gel in the Coomassie stain 5) Pour off the Coomassie Stain. The Coomas
How to destain KimWipes with fresh Destain solution?
Add fresh Destain solution to cover the gel by 3/4 inch (~ 2 cm). 8) Tie Kimwipes in a simple knot and place 4 of them in the Destain solution around the gel. Try to avoid laying the Kimwipes on the gel as this will cause an uneven destaining.
How long to incubate Coomassie gel in destain solution?
Microwave on high power for 40 seconds to 1 minute (until the Destain boils). 10) Incubate the gel in the Destain solution for 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour. 11) Discard the stained Kimwipes and replace with fresh knotted Kimwipes. 12)