Thes procedures are adapted from the procedure at National Diagnostics.
Method 1: Standard (sensitive) Coomassie
This is the classical Coomassie stain, and is highly sensitive, detecting as little as 0.1ug/band. To improve detection a thin gel, run at lower voltage, is preferred as the bands ill be more concentrated under these running conditions.
- Fix gel for 30min to overnight using the fixative solution.
- Stain gel using the fixative solution containing 0.25% Coomassie R
- Stain in a minimal-volume tray
- Cover with ~1.5cm staining solution
- Gently shake 2 - 4hrs, until gel is the same colour as the dye (prior to this, gel will appear as a lighter area)
- Destain 4 - 24hrs in destaining solution. Bands will appear in 1 - 2hrs; destain until background is light-amber or clear
- Store gels in 7% glacial acetic acid.
- 50% methanol
- 10% glacial acetic acid
- 40% ddH2O
- 5% Methanol
- 7.5% glacial acetic acid
- 87.5% ddH2O
Method 1b: Fast Version of Classic Stain
This is a slightly altered version of the standard method, which is completed much more quickly. However, the sensitivity is less with this method and distortions of the gel are a possibility.
- Rinse gel once in ddH2O
- Immerse in staining solution from above protocol, bring to a boil in the microwave (40sec - 1min)
- Shake for 5 - 10min
- Rinse 2X in ddH2O
- Cover gel with 2cm destain solution from above protocol
- Knot 4 kimwipes together and place around gel in destain - DO NOT PLACE ON GEL
- Bring to a boil on microwave (40sec - 1min).
- Incubate on shaker an additional 10min
- Discard kimwpies and replace with 4 fresh knotted kimwipes
- Incubate on shaker an additional 10min to over night. A second round of microwaving can be used to speed the process.
Method 2: Rapid Coomassie Blue R-250
This method is faster than the classical method, but is 2 - 5 times less sensitive.
- Fix gel in 25% isopropyl alcohol, 10% glacial acetic acid (remainder ddH2O), 30 - 60 minutes.
- Stain gel in 10% Acetic Acid in ddH2O, containing 60 mg/L of Coomassie Blue R-250.
- Bands will appear in 30 minutes. Allow staining to proceed until desired band intensity is reached.
- No de-stain is required.
Method 3: Rapid Coomassie Blue G-250
This method is the easiest, but is the least sensitive. Simply soak gel in staining solution; bands should appear in 15 min, and increase in intensity over several hours. Staining solution is stable for several weeks at room temp.
- dissolve 0.2g dye in 100 ml ddH2O (this will require warming to approximately 50°C).
- Cool and add 100 ml 2N H2S04.
- Incubate at room temperature 3 hours to overnight, then filter.
- To filtered solution, CAREFULLY add 22.2 ml 10N KOH
- Then add 28.7g TCA. Allow to stand > 3 hours
- Filter again if necessary to obtain an amber-brown solution without blue precipitate.
Method 4: Coomassie Staining of PVDF Membrane
- After transfer wash blot in ddH2O, 2-3 X 5min
- Stain PVDF membrane with 0.1% Coomassie R-250 in 50% methanol for 15 min.
- Destain with 40% methanol, 10% acetic acid. Change destain as needed.
- Rinse with ddH2O and dry
Method 2 (rapid)
- Wash 1X 5min in TBS-T
- Stain using 50% methanol, 7% acetic acid, 0.1% coomassie R-250; on rocker, 5min.
- Destain with 50% methanol, 7% acetic acid, ~5min (until solution is saturated with dye)
- Replace with 90% methanol, 1% acetic acid; agitate by hand until bands are desired colour - DO NOT OVER-RINSE
- Wash with water and dry
Method 3 (sequencing)
From <ref name="Biosync">http://www.biosyn.com/faq.aspx?qid=310</ref>.
- Rinse membrane 2-3X in ddH2O, 5 min per rinse
- Soak in 100% methanol, 10 min
- Stain PVDF membrane with 0.1% Coomassie R-250 in 40% methanol for no longer than ONE MINUTE usually 15 to 20 seconds will suffice
- Destain with 40% methanol, change destain as needed
- Wash 5x ddH2O
- Dry membrane