NAME: SHAHID JAMAL
NEPTUNE CODE: GDWKUX
AIM: To perform SDS-PAGE
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) is a technique used in biochemistry, genetics and molecular biology to separate protein according to their molecular weight.
The purpose of SDS-PAGE is to separate protein according to their molecular weight .As proteins are amphoteric in nature their net charge therefore can be determined by the pH of the medium in which they are suspended. Therefore at a given pH and under non denaturing conditions the electrophoretic separations of proteins is determined by both size and charge of molecules. As proteins are high molecular weight molecules they need a porous gel to get separated that is why polyacrylamide is used.
Acrylamide solution,gel loading buffer,running buffer,staining and destaining solution,ammonium persulphate,molecular weight marker,protein samples, 10% SDS, 1.5 Tris-HCL(pH=8.8),0.5m Tris-HCL(pH=6.8),50% glycerol,10%,?-mercapthanol,0.03% Bromophenol blue.
Tank with lid, combs, glass plates with spacer, short plates, casting frames, sample loading guide, casting stands.
1.Wash the glass plates with hot water using a detergents
2.Wash off all detergents with plenty of hot waters and after this stands glass plates in vertical position to dry(Precaution: always wear gloves to avoid fingerprints on the glass plates)
PREPARATIONS OF SDS-Polyacrylamide gel
we prepare a two types of gel that is stacking gel with large pore on the top and resolving gel with small pore
Sample and electrophoresis buffer
STEPS OF ELECTROPHORESIS
1.Making of separating gel
-Hold two glass plates in the casting frame on the casting stands
-Pipette appropriate amount of prepared separating gel solution into the gap between the glass plates.
-To make the top of the separating gel horizontal, fill the water in the gap of the glass till it overflows.
-Now after this keep the gel for 20-30 min to be gelate
2.Making of stacking gel
-Before pipeting the stacking gel solution discard the water and we can see the separating gel clearly
-Now start pipeting the stacking gel solution until it start overflows
-Insert the comb into the gel for making the well withoiut trapping air under the teeth of combs
-Again wait for 20-30 minutes to become it gelatinised
3.Before taking out the comb make sure a complete gelation of the stacking gel.
4.Take out the glass plates from the casting frame and set them in the buffer tank
5.Pour the running buffer into inner and outer chamber.
6.Now prepare your protien sample and mix the sample with sample buffer.
Heat them at 95?c for 5 minutes
7.Start loading the prepare sample into well and make sure not to overflow.
Don’t forget loading protien marker into the first lane
8.After this now cover the top and connects the anode.Set the appropriate volt and run the electrophoresis
The total running time 16h for 120v at 4?c
9.Dye the gel using CBB-R250
1 2 3 4 5 6 7 8 9 10
Lane 6 is unknown protien by comparing with the lane 10 of the ladder(10kDa-180kDa).I concluded that my protien size is ?55kDa.
Protien staining in the gel creates a documentable banding pattern of the various protiens. Glycoprotien have differential level of glycosylation and absorb SDS more unevenly at the glycosylation site resulting in broader and blurred bands.
SDS-PAGE is used in the combination with the western blot for the determination of the presence of a specific protien in a mixture of protiens or for the analysis of post translational modification.