assay protocol

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SDS-PAGE, with full name of sodium dodecyl sulfate polyacrylamide gel electrophores, is the most widely used technique to separate proteins from complicated samples of mixture, plays key roles in molecular biology and wide range of subfield of biological research. Being present a electricity, proteins migerate towards the negative anode inside the poly-acrylamide gel under denaturing conditions. In SDS-PAGE, the detergent SDS and a heating step determine that the electrophoretic mobility of a single kind of protein is only affected by its molecular weight in the porous acrylamide gel.

SDS PAGE Preparation:

An intact SDS PAGE electrophoresis system should include: a tank, lid with power cables, electrode assembly, cell buffer dam, casting stands, casting frames, combs(usually 10-well or 15-well), and glass plates (thickness 0.75mm or 1.0mm or 1.5mm). (Bio-rad brand one is recommended)

The SDS PAGE gel in a single electrophoresis run can be divided into stacking gel and separating gel. Stacking gel (acrylamide 5%) is poured on top of the separating gel (after solidification) and a gel comb is inserted in the stacking gel. The acrylamide percentage in SDS PAGE gel depends on the size of the target protein in the sample. (details shown below)

Acrylamide % M.W. Range
7% 50 kDa - 500 kDa
10% 20 kDa - 300 kDa
12% 10 kDa - 200 kDa
15% 3 kDa - 100 kDa

Volumes of stacking gel and separating gel differ according to the thickness of gel casting:

Thickness of the gel Vol. of stacking gel Vol. of separating gel
0.75mm 2ml 4ml
1.0mm 3ml 6ml
1.5mm 4ml 8ml

For a 5 ml stacking gel:

H2O 2.975 ml
0.5 M Tris-HCl, pH 6.8 1.25 ml
10% (w/v) SDS 0.05 ml
Acrylamide/Bis-acrylamide (30%/0.8% w/v) 0.67 ml
10% (w/v) ammonium persulfate (AP) 0.05 ml
TEMED 0.005 ml

For a 10ml separating gel:

Acylamide percentage 6% 8% 10% 12% 15%
H2O 5.2ml 4.6ml 3.8ml 3.2ml 2.2ml
(30%/0.8% w/v)
2ml 2.6ml 3.4ml 4ml 5ml
1.5M Tris(pH=8.8) 2.6ml 2.6ml 2.6ml 2.6ml 2.6ml
10% (w/v)SDS 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml
10% (w/v) ammonium persulfate (AP) 100μl 100μl 100μl 100μl 100μl
TEMED 10μl 10μl 10μl 10μl 10μl

Note: AP and TEMED must be added right before each use.

5X Sample buffer (loading buffer):

10% w/v SDS
10 mM Dithiothreitol, or beta-mercapto-ethanol
20 % v/v Glycerol
0.2 M Tris-HCl, pH 6.8
0.05% w/v Bromophenolblue

Make sure your target protein dissolved in the liquid phase, and no inappropriate ingredients present (e.g. guanidine hydrochloride can interact with SDS and cause precipitation) Generally, to treat your unprepared sample, you can use sonicator, lysis buffer or both to sufficiently make your target protein released, and centrifuge to make supernatant and pellet separated.

1x Running Buffer:

25 mM Tris-HCl
200 mM Glycine
0.1% (w/v) SDS

(Approximately vol. of less than 1 liter is needed depending on the type of your electrophoresis system.)

SDS PAGE Protocol:

1. Make the separating gel:

Set the casting frames (clamp two glass plates in the casting frames) on the casting stands.

Prepare the gel solution (as described above) in a separate small beaker.

Swirl the solution gently but thoroughly.

Pipet appropriate amount of separating gel solution (listed above) into the gap between the glass plates.

To make the top of the separating gel be horizontal, fill in water (either isopropanol) into the gap until a overflow.

Wait for 20-30min to let it gelate.

Make the stacking gel:

Discard the water and you can see separating gel left.

Pipet in stacking gel untill a overflow.

Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate.

2. Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.

3. Prepare the samples:

Mix your samples with sample buffer (loading buffer).

Heat them in boiling water for 5-10 min.

4. Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes.

5. Set an appropriate volt and run the electrophoresis when everything's done.

6. As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 1 hour for a 120V voltage and a 12% separating gel. For a separating gel posessing higher percentage of acylamide, the time will be longer.

Note: Various factors affect the properties of the resulting gel.

  • Higher concentration of ammonium persulfate and TEMED will lead to a faster gelation, on the other hand, a lower stability and elasticity.
  • The optical temperature for gel gelation is 23°C-25°C. Low temperature will lead to turbid, porous and inelastic gels.
  • The pH is better to be neutral and the gelation time shoud be limited in 20-30 min.

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