Coomassie Staining

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Thes procedures are adapted from the procedure at National Diagnostics.

Contents

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.

Protocol
  1. Fix gel for 30min to overnight using the fixative solution.
  2. 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)
  3. Destain 4 - 24hrs in destaining solution.  Bands will appear in 1 - 2hrs; destain until background is light-amber or clear
  4. Store gels in 7% glacial acetic acid.
Recipes
Fixative
  • 50% methanol
  • 10% glacial acetic acid
  • 40% ddH2O
Destain
  • 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.

  1. Rinse gel once in ddH2O
  2. Immerse in staining solution from above protocol, bring to a boil in the microwave (40sec - 1min)
  3. Shake for 5 - 10min
  4. Rinse 2X in ddH2O
  5. Cover gel with 2cm destain solution from above protocol
  6. Knot 4 kimwipes together and place around gel in destain - DO NOT PLACE ON GEL
  7. Bring to a boil on microwave (40sec - 1min).
  8. Incubate on shaker an additional 10min
  9. Discard kimwpies and replace with 4 fresh knotted kimwipes
  10. 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.

  1. Fix gel in 25% isopropyl alcohol, 10% glacial acetic acid (remainder ddH2O), 30 - 60 minutes.
  2. Stain gel in 10% Acetic Acid in ddH2O, containing 60 mg/L of Coomassie Blue R-250.
  3. Bands will appear in 30 minutes. Allow staining to proceed until desired band intensity is reached.
  4. 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.

Staining Solution:
  1. dissolve 0.2g dye in 100 ml ddH2O (this will require warming to approximately 50°C).
  2. Cool and add 100 ml 2N H2S04.
  3. Incubate at room temperature 3 hours to overnight, then filter.
  4. To filtered solution, CAREFULLY add 22.2 ml 10N KOH
  5. Then add 28.7g TCA. Allow to stand > 3 hours
  6. Filter again if necessary to obtain an amber-brown solution without blue precipitate.


Method 4: Coomassie Staining of PVDF Membrane

Method 1

From [1][2].

  1. After transfer wash blot in ddH2O, 2-3 X 5min
  2. Stain PVDF membrane with 0.1% Coomassie R-250 in 50% methanol for 15 min.
  3. Destain with 40% methanol, 10% acetic acid.  Change destain as needed.
  4. Rinse with ddH2O and dry


Method 2 (rapid)

From [3].

  1. Wash 1X 5min in TBS-T
  2. Stain using 50% methanol, 7% acetic acid, 0.1% coomassie R-250; on rocker, 5min.
  3. Destain with 50% methanol, 7% acetic acid, ~5min (until solution is saturated with dye)
  4. Replace with 90% methanol, 1% acetic acid; agitate by hand until bands are desired colour - DO NOT OVER-RINSE
  5. Wash with water and dry


Method 3 (sequencing)

From [4].

  1. Rinse membrane 2-3X in ddH2O, 5 min per rinse
  2. Soak in 100% methanol, 10 min
  3. 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
  4. Destain with 40% methanol, change destain as needed
  5. Wash 5x ddH2O
  6. Dry membrane


References

  1. http://world-2dpage.expasy.org/swiss-2dpage/docs/protocols/protocols.fm9.html
  2. http://www.ncbi.nlm.nih.gov/pubmed?term=1281090
  3. http://www.ecu.edu/cs-dhs/biochemistry/upload/PVDF-Coomassie-Protein-Staining.pdf
  4. http://www.biosyn.com/faq.aspx?qid=310
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