BasicProteinChemistryTechniques

實驗概要

Basic Protein Chemistry Techniques

實驗步驟

Coomassie Blue Stain:  (for gels) 
1) Combine 225 ml Methanol with 225 ml ddH2O. 
2) Add 0.5 grams of Coomassie Blue. 
3) Just before use, add 50 ml acetic acid to 10% final concentration. 
4) Stain for 2 hours. Staining time can be shortened by microwaving; ~50 seconds at medium power. 
5) After staining, pour off the stain into a bottle marked Used Stain.  Stain can be re-used once, but fresh 10% acetic acid must be added).


Coomassie Blue Destain:  (for gels) 
1) Combine 25 ml of Methanol with 437.5 ml ddH2O. 
2) Just before use, add 37.5 ml acetic acid to 7.5% final concentration. 
3) Destain, plus sponges (pieces of foam packing) for a couple of hours.  It may be necessary to use more than one change of destain.   Microwaving can be used in this stage also, but the details have not been determined.

  
Coomassie Blue Stain:  (for membranes) 
1) Combine 50 ml of Methanol with 50 ml of ddH2O. 
2) Add 0.1 g of Coomassie Blue. 
3) Stain by pouring a sufficient amount to cover the membrane in a petri dish. 
4) Wash the fluid gentle back and forth over the membrane.  Allow it to be immersed for one to two minutes. 
5) Dispose of stain in a manner similar to that for the gel stain.

Dialysis Tubing Prep 
1) Cut tubing to appropriate lengths and place them in a large beaker. 
2) Add enough 5% NaHCO₃ (add 50 grams per liter of ddH2O) to cover the tubing. 
3) Bring to a rolling boil and allow it to boil for 15 minutes.  The tubing will float so place a beaker of the next smaller size on top of the tubing. 
4) Rinse the tubing with an excess of ddH2O. 
5) Soak the tubing in 2 mM EDTA (add 10 ml 0.2 M to 1 liter ddH2O) for one hour. 
6) Rinse again with an excess of ddH2O. 
7) Store in 0.05% Azide (add 0.005 ml per liter of ddH2O).

To Pour Stacking Gel...
0.4mls  Acrylamide                    
0.4mls  10x Stacking Buffer        
40uls  10% SDS    
3.12mls  ddH20    
2uls  Temed    
34uls  APS   

How to Make 15% Mini-gels
**Important** Due to the fact that Acrylamide is being used, it is extremely important to wear gloves throughout this procedure.
1) Measure out 5.152-ml ddH2O into a sterile 15-ml-tube.
2) Add 4.58-ml Acrylamide. 
3) Next add 1.1-ml 10x Seperating Buffer.
4) Then add 110-ml 10% SDS. 
5) Continue by adding 56-ml APS. 
6) Finally, add 5.6-ml Temed.(Note: Temed is the polymerizing agent so the following steps must be done fairly quickly.) 
7) Use a disposable pipet, homogenize the solution being careful not to cause any bubbles. 
8) Then use the same pipet to fill the glass leaving about 1 inch at the top. 
9) Finally, add 0.5 inch of H2O-saturated butanol on top of the gel. Note: Leave the unused portion of the solution in the 15-ml tube. Use this to determine if your gel is ready to use (when the tube is polymerized, so is your gel).