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SDS-PAGE standard

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:	1234--other--
Percentage of acrylamide:	7.51012.51517.5--other--%
Volume of separation gel:	6--other--ml

Step 1

Setup the SDS-PAGE gel casting system and check for leakage with water.

Step 2

Prepare --other--ml  separation gel (--other--% ) in a disposable tube as follows:

• --other--ml  separation buffer (4X)
• --other--ml  30% acrylamide/bisacrylamiide (29:1)
• --other--ml  dH₂0

Step 3

Only add directly before casting the gel !!

• --other--µl  fresh APS (10%)
• --other--µl  TEMED

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 --other--  gels by mixing in a disposable tube:

• --other--ml  stacking buffer (4X)
• --other--ml  30% acrylamide/bisacrylamide (29:1)
• --other--ml  dH₂0

Step 8

Only add directly before casting the gel!!

• --other--µl  fresh APS (10%)
• --other--µl  TEMED

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 369121518--other--µl  of the protein sample with --other--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

 

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