Principle, operation and result disposal of SDS-PAGE在线视频

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis,

or SDS-PAGE, is a widely-used technique

for separating mixtures of proteins based on their size.

it is a useful tool for assessing protein size and purity.

Successful completion of this technique is an essential first step

for many methods of protein analysis, like Western blot.

This video presents an introduction to SDS-PAGE,

and then demonstrating its step-by-step procedure.

Various experimental parameters,

such as the polyacrylamide concentration and voltage applied to the gel,

are discussed.

Coomassie staining methods is also introduced.

In order to understand the SDS-PAGE technique,

you must first understand its principle components.

SDS or Sodium-Dodecyl Sulfate,

is a detergent with hydrophilic group and negative charge.

PAGE or Polyacrylamide Gel Electrophoresis is a technique

to separate macromolecules according to their electrophoretic mobility.

Proteins with the same molecular weight but with different charges

and in their native form migrates at different velocities through the Gel.

SDS destroy non-covalent bonds in the proteins,

denatures them causing the molecules to linearize

and lose their native shape and adds a negative charge.

After treatment with SDS

the Proteins with the same molecular weight will move at the same velocity.

In order to identify proteins by size,

protein standards of a known size are loaded along with samples

and run under the same conditions.

Installation of vertical slab electrophoresis unit.

Use two clean and dry glass plates to form a vertical slab unit.

Mark a line at glass plate at about 1 cm below the comb.

Take a small beaker

and prepare the separating gel of 10% according to Table.

Add the above separating gel

into the space between two glass plates to the line.

Gently knock the glass to remove bubbles from the gel

and then cover with 0.5-cm-thick water on the gel surface.

Place the glass at room temperature for 30 min.

After gel polymerization is complete, remove the water with filter paper.

Prepare the stacking gel of 5% according to the above table and shake.

Load the gel solution onto the separating gel.

insert the comb into the stacking gel

and place the gel at room temperature for 30 min.

Mix 10 μl of standard protein or protein sample

with sample dissolved solution at same volume,

and then heat it for 5 min in boiling water.

Place it into the gel apparatus.

Fill the middle and outer chamber with electrode buffer

above the top of the wells in the gel.

Remove the comb from a previously prepared gel cassette.

Load 10 μl of sample into well.

Attach power supply. Red to red, black to black.

run gel at 100V.

Turn off the power when bromophenol blue reached into the tanks.

Separate the two glass plates and place the gel into a big dish.

Add adequate staining solution into the dish

and immerse the gel for 1 hour.

Take the gel into a new dish

and wash at least three times with destaining solution.

Destain the gel until the bands are properly seen.