Phosphate amino acid analysis experiment
Phosphate amino acid analysis experiment
It is of interest to identify phosphorylated amino acid residues in proteins. Phosphorylation occurs at serine, threonine, and methionine in proteins, and the identification of labeled phosphorylated amino acids can be facilitated by partial hydrolysis with HCl followed by bidirectional thin-layer electrophoresis.
Operation method
acid hydrolysis
Materials and Instruments
Phosphorylated proteins Move 1. Preparative electrophoresis of radiolabeled phosphorylated proteins on SDS-polyacrylamide gels. Electrotransfer to a PVDF membrane and wash the membrane several times with water without letting it dry out. For more product details, please visit Aladdin Scientific website.
India ink HCl Phosphate amino acid mixture Electrophoresis buffer Ninhydrin
PVDF membranes Ovens Centrifuges Centrifuge tubes Cellulose thin layer chromatography Filter paper Thin layer electrophoresis apparatus
2. Stain the membrane with 30-50 ml of Indian ink stain for 5-10 min and shake gently until bands appear. Radioactive autoradiography can also be performed by wrapping the membrane with plastic film after marking the radioactive or phosphorescent positions.
3. Cut off the membrane containing the target band with a clean blade and rewet the membrane with methanol for 1 min and then with more than 0.5 ml of water. Place in a microcentrifuge tube with a screw cap.
4. Add a sufficient amount of 6 mol/l HCl to submerge the membrane, tighten the cap of the tube, and incubate in an oven at 110°C for 60 min.
5. Cool naturally, centrifuge at high speed for 2 min, transfer the hydrolysate of the liquid phase to a new microcentrifuge tube, and dry under vacuum for 2 h. The liquid phase was then dried for 2 h. The liquid phase was then dried under vacuum for 1 h.6. Add 6-10 μl of water, shake vigorously on a vortex mixer to dissolve the sample, and centrifuge at high speed for 5 min.
7. Dispense 25-50% of the sample into the wells of a 20 cm x 20 cm x 100 μm cellulose thin layer chromatography plate. The sample was dispensed in small portions of 0.25 to 0.5 μl each, and the spot was blown dry with compressed air delivered through a cotton stuffed Pasteur pipette between each dispense.
8. Dispense 1 μl of a non-radioactive phosphate amino acid standard mixture (containing serine phosphate, threonine phosphate and tyrosine phosphate) on top of each sample in 0.25 to 0.5 μl portions as above.
9. In a large glass tray or plastic box, moisten a large piece of blotting paper (with four holes) with pH 1.9 electrophoresis buffer and slowly pour out the excess buffer. Transfer this large blotting paper over the spiked thin-layer chromatographic plate so that the four spiking points on the plate are centered in the four holes of the large blotting paper, press the paper gently to moisten the fibrils and the sample, and remove the paper when the plate is uniformly moistened.
10. Place the thin-layer chromatography plate in the electrophoresis device, and lap the left and right sides of the plate with Whatman 3MM paper at both 0.5 cm edges, and if there are air bubbles, flatten them out. Cover the plate and start electrophoresis. Thin-layer plates containing 4 samples were electrophoresed at 1.5 KV for 20 min in the HTLE 7000 electrophoresis apparatus with double-thick Whatman 3MM paper laps.
Figure 1 Phosphate amino acids separated by first direction electrophoresis at pH 1.9
11. After electrophoresis, remove the thin-layer plate and dry it quickly with a hairdryer for 20 min (without heating).
12. Wet a small piece of blotting paper with pH 3.5 electrophoresis buffer and wet the plate evenly as described in step 10, taking care to avoid placing the paper on top of the sample.
13. Remove the blotting paper and turn the chromatographic plate 90° counterclockwise for electrophoresis in the second direction. If using an HTLE 7 000 electrophoresis machine, electrophoresis is carried out for 16 min at a voltage of 1.3 KV with an electrode buffer of pH 3.5.
14. After electrophoresis, take out the booklet, dry bake it in an oven at 50~80℃ for 20~30 min until it is completely dry, spray 0.25% ninhydrin acetone solution, and then heat it up for 5~10 min to make the phosphoric acid amino acid standard blot.
15. After the addition of radioactive or phosphorescent localization markers to the thin-layer chromatographic plates, the plates are self-developed using a sensitization screen at -70°C for a period of time ranging from overnight to approximately 10 days.
16. After self-development, the positions of the standard amino acids of phosphate, which have been positionally labeled and stained, are traced on a transparent projection film and the chromatographic plate is retained. The radiologically active amino acid phosphate can be identified by comparing the projection film with the photographic film.
Fig. 2 Second direction electrophoretic separation of phosphoamino acids at pH 3.5
Fig. 3 Radioactive autoradiographic pattern of two-way electrophoretic separation