Akipp4 at 16:54, 2 August 2024

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	~~Thes~~ procedures are adapted from the procedure at [http://www.nationaldiagnostics.com/article_info.php/articles_id/80 National Diagnostics].		These procedures are adapted from the procedure at [http://www.nationaldiagnostics.com/article_info.php/articles_id/80 National Diagnostics].

	= Method 1: Standard (sensitive) Coomassie<br> =		= Method 1: Standard (sensitive) Coomassie<br> =

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Thes procedures are adapted from the procedure at [http://www.nationaldiagnostics.com/article_info.php/articles_id/80 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. 

===== Protocol =====

#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. 

===== 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. 

#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. 

===== Staining Solution: =====

#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 =

===== Method 1 =====

From <ref>http://world-2dpage.expasy.org/swiss-2dpage/docs/protocols/protocols.fm9.html</ref><ref>http://www.ncbi.nlm.nih.gov/pubmed?term=1281090</ref>. 

#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) =====

From <ref>http://www.ecu.edu/cs-dhs/biochemistry/upload/PVDF-Coomassie-Protein-Staining.pdf</ref>.

#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 

 

= References =

<references />

Coomassie Staining - Revision history

Akipp4 at 16:54, 2 August 2024

Admin: Created page with "Thes procedures are adapted from the procedure at [http://www.nationaldiagnostics.com/article_info.php/articles_id/80 National Diagnostics]. = Method 1: Standard (sensitive)..."