Biology Protocols

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SDS-PAGE buffers and solutions

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Resolving buffer:

1.5M Tris pH 8.8

0.4% SDS

for 250mL:

Dissolve 46.75g of Tris in 100mL of dH2O, pH slowly to 8.8 with conc. HCl, add 1g SDS and dissolve thoroughly and finally adjust volume to 250mL with dH2O.

Stacking buffer:

0.5M Tris pH 6.8

0.4% SDS

for 250ml:

Dissolve 15.13g of Tris in 100mL dH2O, pH  slowly to 6.8 with conc. HCl, add 1g SDS and dissolve thoroughly and finally adjust volume to 250mL with dH2O.

10% APS:

for 10mL:

Add 1g of ammonium persulphate to 10mL of dH2O, mix until dissolved.  Filter sterilise and aliquot into 1mL fractions and store at -20C.

10X SDS-PAGE Tris-Glycine-SDS running buffer:

for 1L:

Add 30.3g Tris, 144g glycine and 10g SDS to a 1L beaker or measuring cylinder and add 1L dH2O.  Slowly mix the solution until everything is fully dissolved.

3X SDS-PAGE loading buffer:

for 10mL:

Add 2.4mL Tris/HCl pH 6.8, 3mL 20% SDS, 3ml glycerol, 1.6mL B-mercaptoethanol and 6mg bromophenol blue to a universal tube/small volume measuring cylinder and make to 10ml with dH2O.

30% 37.5:1 acrylamide/bisacrylamide solution:

for 1L:

Add 300g of acrylamide, 8g bisacrylamide to a 1L beaker/measuring cylinder and make to 1L with dH2O. Filter and store away from light at 4C.

Coomassie Stain:

for 550mL:

Add 1.25g coomassie brilliant blue, 250mL methanol, 50mL acetic acid to a measuring cylinder and make to 550mL with dH2O.  Filter sterilise. (Limit of detection is to ~0.1-0.5 micrograms of protein per band).

Destain:

for 1L:

Add 100ml acetic acid, 300ml methanol to a measuring cylinder and make to 1L with dH2O.

Separation ranges:

% Gel

M.W. Range

7

50 kDa - 500 kDa

10

20 kDa - 300 kDa

12

10 kDa - 200 kDa

15

3 kDa - 100 kDa

Choose your desired separation range and then use the SDS-PAGE electrophoresis calculator to calculate the volumes required for resolving (7-15%) and stacking gels (generally 4-5%).

Alternatively if using the buffers detailed in the recipe section use the table below to make up 10% gels (a good general concentration):

Resolving Gel (10%) 1 gel 2 gels
30% acrylamide 2.7 mL 5.4 mL
Resolving buffer 2.0 mL 4.0 mL
dH2O 3.25 mL 6.5 mL
ammonium persulphate (10%) 75 uL 150 uL
TEMED 10 uL 20 uL
Stacking Gel
30% acrylamide 375 uL 750 uL
Stacking buffer 625 uL 1.25 mL
dH2O 1.5 mL 3 mL
ammonium persulphate (10%) 20 uL 40 uL
TEMED 5 uL 10 uL

 

 

 

 

 

 

 

 

 

 

Reference:

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.


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