Serum protein polyacrylamide gel electrophoresis.

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1. Master the principle of electrophoresis of polyacrylamide gels.

2. Familiar with the operation process of polyacrylamide gel electrophoresis.

3. Understand the characteristics and application range of polyacrylamide gel electrophoresis.

"Experimental Principles

1. Polyacrylamide gel polymerization principle Polyacrylamide gel (PAG) is a synthetic gel, composed of acrylamide (Acrylamide Acr) and crosslinker methicilline (N, N1-methylene-bis acrylamide, short-form Bis) under the action of a catalyst (perthionamine or nucleolutin), polymerized into a fat family long chain containing alamide-based side chains, adjacent two chains formed by the intersection of the fork bridge with a three-dimensional mesh structure of polyacrylamide gel. To speed up polymerization, tetamine is also added as an accelerator when synthesized into the gel.

polyacrylamide gel has a three-dimensional mesh structure and can act as a molecular sieve. Using it as an electrophoresis support, the separation of the sample depends not only on how much charge each part charges, but also on the molecular size. The size of the gel mesh hole is mainly affected by the total concentration of the gel and the ratio of Acr and Bis. The total concentration of gel-forming substances used in general electrophoresis is 7.5%, which is called the standard gel concentration. If the total glue concentration is changed, the ratio of Acr to Bis should also be changed accordingly.

2. The principle of non-continuous polyacrylamide gel disc electrophoresis According to the type and pH of the buffer in each part of the gel, whether the aperture size is the same, etc., can be divided into continuous system and non-continuous system polyacrylamide gel electrophoresis (PAGE). Continuous system refers to the pH of the buffer in the electrophoresis tank is the same as in the gel, while the semen system is different.

disc electrophoresis is usually carried out in small glass tubes. Overlap the three polyacrylamide gels, which are not exactly the same nature. The upper layer is the sample glue, the middle is the concentrate gel, the gel concentration of these two layers is 3% is the large hole glue, the application of Tris-HCl buffer, its pH6.7.

the lower layer is the separator, the total concentration of this layer gel is 7.5%, is a small hole gel, also with Tris-HCl buffer, pH8.9. The upper and lower electrophoresis tank buffer is Tris-Glycine buffer, pH-8.3. Because of the different aperture of the gel and the pH of the buffer, the concentration effect, charge effect and molecular sieve effect are produced, which improves the sensitivity and resolution of electrophoresis.

(1) concentration effect: No matter which electrophoresis method, in order to get a good separation effect, electrophoresis must be started so that the various components in the sample are on the same running line. After the sample is added, the particles are dispersed and cannot be electrophoresis from the same starting point, but the sample is concentrated into a high concentration of sample thin layer through concentrate, so that the particles start electrophoresis at the same time.

Its engine is the sample glue and concentrate tris-HCl buffer pH=6.7, electrophoresis, HCl all ion is Cl-, while the electrophoresis tank Tris-Glycine buffer pH=8.3, glycine and other electrical points pI=6.0, electrophoresis, glycine only a very small part of the electricity Away from glycine negative ions (Gly-), generallyserum protein points about pI-5.0, also dissoidized as protein negative ions (Pr-), its dissosis is smaller than HCl, bigger than glycine.

after the power is applied, the three ions swim in the positive pole at the same time, the effective swimming rate is Cl->Pr->Gly-, when the electrophoresis begins, cl-swimming fastest (called fast ions), quickly over Pr-, swim to the front, Gly-swim the slowest (called slow ions), swim to the end, Pr-in between, concentrated into a small thin layer of sample.

this concentration can concentrate proteins hundreds of times. Serum protein can only be divided into 5 to 7 components on paper electrophoresis and cellulose acetate film electrophoresis. In the polyacrylamide gel electrophoresis can be divided into 20 to 30 components.

(2) charge effect: protein samples are concentrated at the interface into a narrow thin layer of samples that enter the separation glue. Because the isoelectrons of various protein molecules are different, the buffer in the same pH with different effective charge, so the swimming rate is also different, so the various proteins in order of swimming rate in a disc-shaped protein zone.

(3) Molecular sieve effect: various protein molecules due to different molecular size and composition, so through a certain aperture of the separation of the glue by different friction, the degree of blocking is different, the performance of different swimming rates are separated. Even if the protein has a similar net charge, it is separated by the molecular sieve effect.

" equipment and" reagents

1. Equipment electrophoresis instrument, disc electrophoresis tank, micro-sampler, syringe, 50 ml smallbeech

2. Reagent

(1)30% acrylamide storage liquid: acrylamide 30.0g/gt;, methyl diacrylamide 0.8g/gt;, add water to 100ml, brown bottle 4 oC/gt; save.

(2) catalyst (10% ammonium persulphate): ammonium persulphate 1g/gt; add water to 10 ml, ready for use.

(3) accelerator: tethymethyl ethyl diamine (TEMED)

(4) separation adhesive buffer (pH8.8): take trihydroxymethylamine methane (Tris) 36.3g/-gt;, add 1 mol/L HCl 48ml, and add distilled water to 100ml, 4c/gt.

(5) concentrate buffer (pH6.8): Take 6.0g/gt; Tris, use 1 mol/L HCl 48 ml, and add distilled water to 1000ml, 4C/gt;

(6) electrophoretic buffer (pH8.3): Take 28.8g/gt; glycine, 6.0g/gt; Tris, dissolved, add distilled water to 100ml, 4C/gt; save.

(7) staining fluid: Coomas bright blue R-2500.46g/gt; methanol (can be replaced with waterless ethanol) 110ml, 28ml ice acetic acid, TritonX-100 1ml, dissolved filled water to a total volume of 400ml.

(8) decolorizer (30% methanol, 7% acetic acid): methanol (available instead of waterless ethanol) 110ml, ice acetic acid 28ml, TritonX-100 8ml, filled with water to 400ml.

(9) preservation fluid: 7% ice acetic acid.

(10) sample dilution: concentrate buffer 25 ml added sucrose 10g/gt; and 0.05% bromphenol blue 5ml, and finally added water to 100ml.

"Steps

1. The preparation of the gel

(1) take the glass tubes at both ends that have been flattened with gold and steel sand×0.6cm/gt. Draw a line at 7cm/gt; and 7.5cm/gt; Insert the rubber pad and place it vertically < the test tube > therack.

(2) according to the table to make the separation glue, quickly use a dropper to add it along the pipe wall into the glass tube, to 7 cm/gt;