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Category: Experimental Procedures
Author: Admin eLabJournal

Labels: Western blotting

Materials
 

- Separation buffer (4X)  
- Stacking buffer (4X)  
- 30% acrylamide/bisacrylamide (29:1)  
- 10% ammonium persulfate (APS)  
- Tetramethylethylenediamine (TEMED) 116.24 g mol-1
- Butanol (water-saturated)  
- SDS-PAGE running buffer (1X)  
- SDS-PAGE sample buffer (4x)  
- SDS-PAGE gel casting system  
- SDS-PAGE gel running system  
- Power supply  

Experiment settings

- Number of gels:  
- Percentage of acrylamide:
- Volume of separation gel: ml 

Step 1
Setup the SDS-PAGE gel casting system and check for leakage with water.
Step 2
Prepare ml separation gel ( ) in a disposable tube as follows:
Step 3
Only add directly before casting the gel !!
Step 4
Immediately cast the gel (leave 2-3 cm for the stacking gel) and add 1 ml of butanol (water-saturated)
Step 5
Allow the gel to polymerize for at least 1h
Step 6
Remove the butanol, rinse with some dH20 and remove all liquid with some filter paper
Step 7
Prepare the stacking gel for   gels by mixing in a disposable tube:
Step 8
Only add directly before casting the gel!!
Step 9
Immediately pipet the stacking gel on top of the polymerized separating gel, insert a comb with the appropriate number of wells and allow the gel to polymerize for 1h
Step 10
Place the gels in the running system and fill the system with SDS-PAGE running buffer
Step 11
Mix µl  of the protein sample with µl  of SDS-sample buffer (4X) and boil for 10 min
Step 12
Load the samples on gel and run the gel at 130 V until the blue loading dye reaches the bottom of the gel.
Step 13
Disassemble the running system to take out the gel. Proceed to CBB staining/Silver staining of the gel or Western blotting
Acrylamide is highly neurotoxic!! Prevent any skin contact and always wear gloves!

Based on: Laemmli UK (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Nature 227 (5259): 680-685