The SDS-polyacrylamide gel electrophoresis method measures the relative molecular weight of proteins.

, purpose
to understand the principle of
SDS
-polyacrylamide gel electrophoresis, and learn to use this method to determine the relative molecular weight of
protein
. 2. Principle
Polyacrylamide gel electrophoresis is able to separate different large molecules
compounds
because of the difference in charge and molecular size of these large molecular compounds, if the difference in charge is removed or reduced to a negligible degree, the migration rate of these compounds on the gel is entirely dependent on the relative molecular quality.
SDS, short for sodium dodecyl sulfate, is an anion defiant that binds to protein molecules in proportion to form a negatively charged complex.
Its negative charge far exceeds the original charge of the protein, thus eliminating or reducing the original charge difference between different proteins, so that the electrophoresis migration rate depends only on the molecular size of this factor, according to the standard protein's relative molecular weight of the vector to the migration rate of the standard curve to find the relative molecular mass of the unknown protein.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) can be electrophoresis with a disc, can also be electrophoresis with vertical flat panel, this experiment with the current commonly used vertical flat-panel electrophoresis, the starting point of the sample is consistent, easy to compare.III,
Reagents
and Equipment
(i) Reagents
1, Gel Storage Liquid: Acrylamide (Acr) 29.2g, METHYLENE diacrylamide (Bis) 0.8g, increased steamed water to 100ml. Outsourced tin paper, 4C refrigerator storage, within 30 days of use.
2, separator buffer: 1.5mol/L Tris-HCl, pH8.8.
18.15 Tris (trihydroxy methamethane), plus about 80 ml of heavy steamed water, with 1mol/LHCl to pH to 8.8, diluted with heavy steamed water to the final volume of 100 ml, 4 degrees C refrigerator storage.
3, concentrate buffer: 0.5mol/LHCl, pH6.8.
6g Tris, plus about 60ml of re-steamed water, with 1mol/LHCl to 6.8, diluted with heavy steamed water to a final volume of 100ml, 4C refrigerator storage.
4, 10% SDS, stored at room temperature.
5, two types of sample buffer:
(1)2 times reduced buffer (2× reducing buffer).
0.5mol/L HCl, pH6.82.5 ml
glyceglycem 2.0 ml
mass concentration 10% SDS4.0 ml
mass concentration 0.1 % bromphenol blue 0.5 ml
β--based ethanol 1.0 ml
total volume 10 ml
(2) 2 times non-reducted buffer (2×non-reducing buffer).
water 1.0 ml
0.5mol/L HCl, pH6.82.5 ml
glyceer oil 2.0 ml
mass concentration 10 %SDS4.0ml
mass concentration 0.1% bromophenol blue 0.5ml
total volume 10 ml
6, electrode buffer, pH8.3.
Tris3g, glycine 14.4g, SDS1.0g added steamed water 1000ml, 4C refrigerator storage.
7, low relative molecular quality standard protein (Produced in Shanghai), dissolved after opening in 200 μl re-steamed water, plus 200 μl 2x sample buffer (reduced buffer), divided into 20 small officials, -20 degrees C preservation. Pre-use boiling water bath 3 to 5min its relative molecular mass (Mr) is as follows: standard protein Mr
rabbit
phosphorylation
enzyme B97 400
nu
serum
albumin 66 200
rabbit creatin 43 0 00
Bovine carbonate enzyme 31,000
pancreas
protease
inhibitor 20 100
egg clear lysomalase 14 400
8, mass concentration of 10% ammonium persulphate: this solution needs to be pre-use preparation.
9, dyeing liquid: 0.25g Kaomas bright blue R250, add 91ml 50% methanol, 9ml ice acetic acid.
10, decolorizer: 50 ml methanol, 75 ml ice acetic acid mixed with 875 ml of re steamed water.
11, relative molecular mass to be tested.
.