Experimental reagent

1. 5x sample buffer (10ml): 0.6ml 1mol/L Tris-HCl (pH 6.8), 5ml 50% glycerol, 2ml 10% SDS, 0.5ml mercaptoethanol, 1ml 1% bromophenol blue, 0.9ml Distilled water. It can be stored at 4 ° C for several weeks or at -20 ° C for several months.

2. Gel stock solution: In the fume hood, weigh 30g of acrylamide and 0.8g of methyl bisacrylamide. After adding distilled water, dissolve it to 100ml. Filtered and placed in a brown bottle, stored at 4 ° C, generally can be placed for 1 month.

3. pH 8.9 separation gel buffer: Tris 36.3g, add 1mol / L HCl 48ml, add 80ml of distilled water to dissolve, adjust pH 8.9, dilute to 100ml, 4 ° C preservation.

4. pH 6.7 concentrated gel buffer: Tris 5.98g, add 1mol / L HCl 48ml, add 80ml of distilled water to dissolve, adjust pH 6.7, dilute to 100ml, 4 ° C preservation.

5. TEMED (tetraethyl ethylenediamine) stock solution

6.10% ammonium persulfate (prepared freshly with distilled water)

7. pH8.3 Tris-glycine electrode buffer: Weigh 1.6 g of Tris, 28.8 g of glycine, add about 900 ml of distilled water, adjust pH to 8.3, and dilute to 1000 ml with distilled water. Store at 4 ° C and dilute 10 times before use.

8. Coomassie Brilliant Blue G250 staining solution: Weigh 100mg Coomassie Brilliant Blue G250, dissolve in 200ml of distilled water, slowly add 7.5ml of 70% perchloric acid, and finally make up the water to 250ml, stir for 1 hour, filter with small hole filter paper.

Laboratory equipment

Electrophoresis apparatus, electrophoresis tank, water bath, shaker.

Experimental procedure

Sample preparation

Protein samples were mixed with 5X sample buffer (20 ul + 5 ul) in an Eppendorf tube. Heat at 100 ° C for 5-10 min, take the supernatant and sample.

2. Preparation of separation gel and concentrated gel

1) Wash the glass plate, sample comb, and Spacer with detergent, rinse several times with ddH2O, wipe with ethanol, and dry;

2) Add Spacer between the two washed glass plates, and install the glass plate according to the instructions of Bio-Rad Mini II/III;

3) Prepare 8.0 ml of 10% separation gel in the following volume and mix;

ddH2O 3.0 ml

1.0 mol/LTris-HCl pH=8.8 2.1 ml

30% Acr-Bis 2.8 ml

10% SDS 80 ul

10% AP 56 ul

TEMED 6 ul

4) Pour the separation glue between the glass plates, immediately cover a layer of re-distilled water, and the glue can be polymerized after about 20 minutes;

5) Prepare 3.0 ml of 6% concentrated gel in the following volume and mix; ddH2O 1.0 mol/LTris-HClpH=6.8 30% Acr-Bis

2.0 ml 400 ul 600 ul

10% SDS 10% AP TEMED

36ul 24ul 4ul

6) Pour the upper layer of distilled water, blot the filter paper, fill the concentrated gel, and insert the sample comb;

7) Install the electrophoresis system, add the electrode buffer, and load 20 μl;

8) When the voltage is 200V, when bromophenol blue just runs out of the separation gel, the electrophoresis is stopped, which takes about 45 minutes to 1 hr.

9) Remove the rubber plate, remove the glue into the staining solution, and dye at room temperature for 1~2 hr; add the decoloring solution, put it on the 80 rpm decolorizer shaker, and replace the decolorization solution every 20 minutes (10 ml glacial acetic acid; 45 ml ethanol) ; 45 ml distilled water) until completely removed.

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