Preparation of acrylamide solutions for casting of gels

Standard Protocol for Casting SDS PAGE Gels

Solution of Acrylamide and N, N'-Methylenbisycrylamide 

Cat. No. Composition
Acrylamide:Bis
% C % T Applications
10679 19:1 5.0 40 DNA Sequencing
10680 29:1 3.3 40 IEF, SDS PAGE
10681 37.5:1 2.6 40 SDS PAGE
10687 29:1 3.3 30 IEF, SDS AGE
10688 37.5:1 2.6 30 SDS PAGE

 

The SERVA Acrylamide/Bis solutions are applicable to all PAGE methods. Acrylamide/Bis solution 29:1 is also suitable for preparation of a gel according to Schägger and Jagow 1). The amount of gel solutions (20 ml) is sufficient for 2 vertical slab mini gels (100 mm x 80 mm x 1,4 mm).

Product
For final concenration of gel (% T)
Seperating gel (10 ml) 
  
 Stacking gel 
(10 ml)
7 %
10 %
12.5 %  
15 %
5 %
30 %*
40 %*
30 % 
40 % 
30 % 
40 % 
30 % 
40 % 
30 % 
40 %
Acrylamide/Bis
Solution %*
2.33 ml
1.75 ml
3.33 ml 
2.50 ml 
4.17 ml
3.13 ml
5.00 ml
3.75 ml
1.67 ml
1.25 ml
Seperating gel
buffer 4 x
2.50 ml
2.50 ml
2.50 ml
2.50 ml
2.50 ml
2.50 ml
2.50 ml
2.50 ml
— 
— 
Stacking gel
buffer 4 x
— 
— 
—  
—  
—  
—  
—  
—  
2.50 ml
2.50 ml
dist. H2O
5.07 ml
5.65 ml
4.03 ml
4.90 ml
3.29 ml
4.27 ml
2.46 ml
3.65 ml
5.79 ml
6.15 ml
SDS, 10 % Solution
(Cat. No. 20763)
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
TEMED
(Cat. No. 35925)
0.015 ml
0.015 ml
0.015 ml
0.015 ml
0.015 ml
0.015 ml
0.015 ml
0.015 ml
0.015 ml
0.015 ml
APS, 10 % Solution
(Cat. No. 13375)
0.03 ml
 0.03 ml
0.03 ml
0.03 ml
0.03 ml
0.03 ml
0.03 ml
0.03 ml
0.03 ml
0.03 ml
 
*The composition of the acrylamide/bis solution (acrylamide content: Crosslinker) depends on the application.
For standard SDS-PAGE, we recommend the Acrylamide/Bis Solution 29:1 (cat. no. 10680) or 37.5:1 (cat. no. 10681).

 

Required Buffers

Seperation gel buffer, 4x (1,5 M Tris/HCI pH 8.8)

• Dissolve 18.2 g Tris (cat. no. 37190) in water (bidest.)
• Adjust pH 8.8  (approx. 2.8 ml HCl, 32 %)


Stacking gel buffer, 4 x (0,5 M Tris/HCI pH 6.8)

• Dissolve 6.1 g Tris (cat. no. 37190) in water (bidest.) 
• Adjust pH 6.8  (approx.  4.4 ml HCl, 32 %)

 

Preparation

  1. Mix acrylamide/bis solution, buffer and water in separate beakers.
  2. Deaerate the solutions briefly (1 to 3 min ad vacuo).
  3. Add to separating gel solution: 10 % SDS solution (w/v in water), TEMED and APS solution (w/v 10 % of ammonium persulfate), gently swirl to mix without incorporating air into the mixture.
  4. Casting the separating gel: fill slab gel sandwich with separating gel solution up to approx. two thirds. The remaining volume for the stacking gel should refer to a separation distance of 1.5-fold of the depth of the wells of the comb to be used. Overlay with isopropanol immediately (0.1 ml), let polymerize (approx. 15 - 20 min).
  5. Add 10 % SDS in water to the stacking gel solution, then TEMED and 10 % ammonium persulfate solution (APS), swirl gently to mix without incorporating air into the mixture.
  6. Casting the stacking gel: fill up the slab gel sandwich to the top and let polymerize.
Literature:
1) Schägger H. & von Jagow, U. (1987) Anal. Biochem. 166, 368-379 2.
2) Laemmli, U.K. (1970) Nature 227, 680-685.

Top
Close window
Product of the Month February:
Protein Quantification Assay Kits

Fast and Reliable Protein Quantification

 → This way to the Special Offer

SERVA InfoMail - Get the latest information

Close window

When subscribing to »SERVA InfoMail« you will receive information about the latest product release, special promotions, new jobs at SERVA and more. This service is, of course, free of charge.
You may remove your name any time from the list of recipients.

Quick Order

Close window

Using the Quick Order field, you can add a product to your shopping cart with just one click. All you have to do: enter the Cat.No. as shown in our catalog in the format xxxxx.yy and click the Go!-Button.