G250 Staining and Equal Sample Loading of Protein Samples

 Equal sample loading of protein samples

Protocol  

• Take 20 µl protein sample and mix with 6.66 µl of 4x reducing buffer (laemmli buffer) 
• Should be adjusted to your desired volume (general formular: V/3 if reducing buffer is 4x)
• Vortex the samples 
• Heat at 95° C for 5-10 min 
• Centrifuge samples 
• Load samples and marker on gel 
• Stack gel for 15 min at 90 Volts 
• Separate samples for 45 min - 1 hour at 130 - 150 Volts  
• Put gel into G250 Coomassie and incubate over night on a shaker 
• You can also heat the gel in a microwave until it cooks and stain for 1 hour (to stain fast) 
• Next day remove Coomassie and add destain solution 
• Destain several rounds until protein bands appear 
• Destaining can be improved by heating the gel in microwave 
• Analyse gel 

Recipes: 

G-250 Coomassie: 

Chemical                     Final Concentration 

G-250                          0.5%

Methanol                     40%

Acetic Acid                 10%

Water                           to 100% 

Destain Solution: 

Chemical                     Final Concentration 

G-250                             0%
Methanol                      40%

Acetic Acid                  10%

Water                            to 100%