Technical principle
SDS polyacrylamide gel electrophoresis, is introduced in polyacrylamide gel system SDS (sodium dodecyl sulfate), SDS and degeneration of polypeptide, protein and negatively charged, because the peptide in combination with the amount of SDS almost always proportional to the molecular weight of peptides and had nothing to do with, so the SDS peptide compounds in propylene amide gel electrophoresis migration rate is only related to the size of the peptides, in saturated state, the peptide can be combined with 1.4 g detergent per gram.
When the molecular weight between 15 kd and 200 kd, the migration rate and molecular weight of protein logarithm linear relationship, conform to the type: logMW = K - bX, type: MW as molecular weight, X for mobility, K and b are constant, if the mobility of standards of known molecular weight protein on molecular weight of logarithmic mapping, a standard curve can be obtained, and the unknown protein electrophoresis under the same conditions, according to its electrophoretic mobility of molecular weight on the standard curve can be obtained.
The experimental items
Protein samples, SDS - PAGE loading buffer, PBS, Tris - Hcl (PH 8.8), Tris - Hcl (PH 6.8), 30% acrylamide, 10% SDS, 10% persulfuric acid, 5 * SDS - PAGE electrophoresis buffer, coomassie bright blue r-250 dye, decolorization fluid.
The experimental steps
I. Gel configuration
1.The preparation of the separation glue
(1) find the supporting glue board, wipe clean with the mirror paper, and fix it on the glue binder.
Then the ionic water test was used.
(2) if the rubber sheet does not leak, make the separation gel.
In turn, mix water, 30% acrylamide, 1.5m Tris - Hcl (PH 8.8), 10% SDS, 10% persulphate and add to clean small beaker, mix well (1mm glue board, usually with a piece of glue with 5ml.
The concentration of the gel is generally 12 %, but the concentration depends on the situation.
The smaller the protein, the greater the concentration of the adhesive.
(3) remove the water from the rubber sheet and drain the water with the filter paper (do not plug the filter into the bottom of the rubber sheet);
(4) add TEMED in the beaker and mix well.
(TEMED can solidify, so add);
(5) grouting: the mixed separation glue is gently under the edge of the rubber plate to prevent bubbles;
(6) use water to flush the glue (fill it with water, and add water slowly when adding water to prevent it from blowing up).
2.The preparation of concentrated glue
(1) the separation gel forms a clear line in the middle of the water, that is, the separation gel solidifies.
Subsequently, the preparation of concentrated adhesive, in turn, water, 30% acrylamide, 1.0 M Tris - Hcl (PH 6.8), 10% SDS, 10% persulphate to clean small beaker;
(2) remove the water from the top layer of the gel and suck up the moisture with the filter paper (try not to insert the filter paper);
(3) add TEMED in small beaker and mix well;
(4) glue.
Then insert the comb;
(5) when the gluing machine is inverted, the adhesive will not be flowing out, or it can be clearly seen that the glue surface between the two teeth in the comb is obviously concave, indicating that the concentrated glue solidifies.
The solidification of plastic board into electrophoresis groove, and join in slot on electrophoresis sds-page electrophoresis buffer, soak glue for a period of time, the longer the better (prevent comb is linked together with glue, or plastic shrinking due to humidity);
(6) when the sample is taken, the comb will be pulled out and the action should be light, preventing the tooth from being pulled out.
II. SDS - PAGE electrophoresis
1. Tuning: remove the comb from the SDS glue and adjust the position of the teeth with the small needle (make them all parallel).
2. Sample: use 20 pieces of the small gun of the L, and absorb the boiled sample 10, and aim at the glue hole. Be careful not to point the sample outside.
Use the appropriate molecular weight protein marker.
3. Electrophoresis: the electrophoresis is positive and negative, turn on the power.
Concentrate: voltage low 100V
Separation adhesive: voltage high 150V.
4. Unglue: all the blue in the glue will go out, and the electrophoresis will stop, usually about 1-2 hours.
Remove the rubber sheet from the meter and use the blade to prise open the rubber sheet.
5. Staining: put in the staining of coomassie bright blue r-250 dyeing fluid;
5. Decoloration: the overnight staining SDS - PAGE gel is put into the decoloration solution until the background is white.
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