Why don't I send you a picture?
Solution:
(1) transfer buffer: weigh 2.9g glycine, 5.8g Tris base, 0.37 SDS and 200ml methanol, add deionized water to 1000ml, and do not add SDS when the protein molecular weight is small.
(2) PBS-T: NaCl 8.0g，KCl 0.2g，KH2PO4 0.2g，Na2HPO4？ 12H2O 2.9g, dissolved in 800ml deionized water, adjusted the PH to 7.4 with hydrochloric acid, and finally dissolved to 1000ml, and added 0.05%Tween20.
(3) Sealing solution: 5% skimmed milk is dissolved in PBS.
(4) Amino black dye solution: 0.2% amino black is dissolved in 7% acetic acid.
(5) Decolorization solution of amino black: 30% methanol, 10% glacial acetic acid solution.
SDS-PAGE related solutions
(1). Electrode buffer:
Three: 6 grams
Glycine: 28.8g.
(10%) sodium dodecyl sulfate: 10 ml.
Add 1000 ml H2O, and the pH value is 8.3.
(2). Separation gel buffer: 100ml:
Three: 36.6 grams
1N hydrochloric acid: (about 48 ml): PH: 8.9.
Add water to: 100 ml.
(3). Concentrated rubber buffer:100 ml;
Three: 5.98 grams
1N HCL: (about 48ml): PH:
Add water to: 100 ml.
(4) Gel storage solution: 100 ml:
ACR:30 grams
Double: 0.8 g
Add water to: 100 ml.
(5).4x load buffer:
2-Me: 20% 2ml
Sodium dodecyl sulfate: 8% 0.8g.
Glycerol: 40% 4ml.
Bromophenol blue: 0.4% 0.04g.
Tris-CL (ph6.8) 240mM 2.5ml (concentrated gel buffer)
Add1.5ml H2O.
Total:10ml
(6) Dyeing solution (rapid dyeing formula, dyeing 1 hour)
0. 1% coomassie blue, 10% acetic acid, 45% methanol
Coomassie blue R-250 1g
Methanol 450 ml
Water 450 ml
Glacial acetic acid 100 ml
(7). Decolorization solution: 1000ml.
Glacial acetic acid: 75 ml
Methanol: 50ml
Water: 875 ml
Cell lysate
(1) buffer A:25mM Tris pH 7.5.
50 mm KCl
2 mm magnesium chloride
1 mm EDTA
5 mM dithiothreitol (DTT)
(2) Nuclear lysate
Buffer NE:
25mm Tris pH 7,5
0.42 M sodium chloride
1.5mm magnesium chloride
0.5 mm EDTA
1 mM dithiothreitol (DTT)
25% sucrose
0.2% SDS (without immunoprecipitation)
Protease inhibitor was added before cell lysis, and the final concentration was 100 mg? PMSF, 1 mg? L- 1 aprotinin, 2 mg? L- 1 leuptin
Steps: SDS-PAGE electrophoresis.
PAGE glue with different concentrations was prepared according to the glue preparation method. Dyes were placed at the bottom of the separation glue by electrophoresis, and the power supply was cut off. Take off the gel,
Protein imprint
1. After electrophoresis, cut the gel with the protein lane to be transferred, and make a mark in the lower right corner to determine the direction and the front and back.
2. Cut off 1 piece of NC film with the same size as the gel (not larger than the gel), mark it in the lower right corner, and soak it in the transfer buffer for about 5 minutes.
3. Cut 8 Zhang Xinhua 1 filter paper and make a mark in the lower right corner, which is slightly the same size as the gel (not larger than the gel).
4. Soak the cut filter paper in the transfer buffer, and install the transfer device from anode to cathode (from bottom to top) in turn: put the bottom electrode flat, put four soaked filter papers on the bottom electrode, align them accurately, and put the NC film on the filter paper to eliminate bubbles. Transfer the SDS-PAGE gel to the transfer buffer, rinse it, lay it flat on the NC membrane, and squeeze out all bubbles with a glass rod. Put four pieces of soaked filter paper on it to discharge bubbles.
5. Put the upper electrode on the interlayer, turn on the power supply, and turn on the power supply according to the gel area 1mA/cm2, and turn on the power for 2 ~ 4 hours. 100 ma, 2 hours.
6. After the transfer, take out the filter paper. Transfer the gel to Coomassie brilliant blue dye tray, and check whether the protein transfer is completed. Cut off the NC film containing molecular weight standard, put it into amino black dye solution for 30 seconds, and the amino black decolorizing solution decolorizes.
7. Wash the NC membrane after protein transfer with distilled water and dry it slightly, then soak it in a sealing solution at 4℃ and seal it overnight, and then wash the membrane with PBS-T buffer for three times, each time 65438±00min.
8. Incubate the membrane with the first antibody for 2 hours at room temperature, dilute the first antibody with PBS-T buffers with different dilutions to find the most suitable concentration of the first antibody, and then wash the membrane with PBS-T buffer for 3 times, each time 10min.
9. Incubate the membrane with HRP-labeled goat anti-mouse IgG (1: 5000) * */h, and wash the membrane with PBS-T buffer for three times, each time 10min.
10. Prepare luminescent matrix (according to 1: 1 mixed matrix solution).
Wash it with 1 1 PBS-T buffer was used to develop color on chemiluminescence substrate for 2 minutes, and the gel imaging system was used to observe and take pictures. (Or develop color in dark place for 0 ~ 15 minutes in DAB chromogenic solution, and wash with water immediately after the color reaction is interrupted by bands).
I'm just giving an example, because only the experiments you come up with are handy. If someone teaches you to do experiments, your ideas will follow others and may be misled by others. What's the point of doing this experiment?