Protein recovery assay
Summary
Recovery test is a type of "control test". When the analyzed sample is complex and not completely clear, a known amount of the measured component is added to the sample and then measured to check whether the added component can be quantitatively recovered, in order to determine whether there is a systematic error in the analytical process. The result is often expressed as a percentage and is called "percent recovery", or "recovery rate" for short.
Operation method
basic program
Materials and Instruments
Protein
SDS DTT Decolorizing Solution Staining Solution
Electrophoresis Dialysis Membrane
Move
1. The gel was immersed in staining solution at room temperature for 15~20 min with slow shaking, and then transferred to decolorizing solution at 4℃ for 2~3 h with gentle shaking.
2. Cut off the stained gel strips and soak them in water at 4°C for 2~3 h, during which time the water was changed several times. Then each strip of gel was separately transferred into a Petri dish, 10 ml of water was added to cover it, and they were mashed into pieces of about 1 mm3, and then the water was sucked up by thousands with a Pasteur pipette.3. Cover the bottom end of the elution cell of the electroelution apparatus with a sheet of Spectrapor 6 dialysis membrane and tighten the cap.
Figure 1. Elution Cell
4. Transfer the mashed gel into the rough end of the elution chamber and cover with soaking buffer. On top of the soaking buffer, add a layer of elution buffer just above the horizontal tunnel to eliminate air bubbles.5. Place the elution chamber within the elution cell, add the eluent just above the drain outlet of each electrode tank, and add the rest of the liquid to the small mixing cell.6. Connect a two-channel peristaltic pump and adjust the flow rate so that the solution drips slowly from the mixing cell back into the elution cell. Remove all air bubbles from under the dialysis membrane in the elution chamber using an elbowed Pasteur pipette, being careful not to puncture the membrane.7. Soak the gel pieces for 3-5 h. Attach the electrodes, taking care to connect the cathode to the side of the stripping chamber with the gel pieces. 50 V constant voltage for 12-16 h.8. Turn off the power, disconnect the electrode, and replace the eluent in the elution tank with dialysate. Reconnect the electrodes at a constant voltage of 80 V for 12 to 24 minutes,9. Turn off the power, disconnect the electrodes and turn off the peristaltic pump. Remove the elution chamber from the tank, aspirate the buffer above the blue protein layer in the collection cell containing the eluted proteins, and mix the remaining protein-containing liquid with a 50 μl syringe with a flat tip needle.10. Pipette the protein solution into a microcentrifuge tube, wash the collection cell with 50 μl of dialysate and combine in the protein solution.11. The liquid is lyophilized in a Speedvac evaporator and the sample can be stored at -20°C.
Figure 2. Elution apparatus and elution cell
12. Samples were reconstituted with 100 μl of water and precipitated with 50:50 methanol/acetone. Proteins were precipitated by microcentrifugation at -20°C overnight.13. Take 5-10% of the sample and use the microgel of Laemmli gel system to separate the degree of protein elution, molecular weight and recovery, and the protein can be detected by the method of Thomas Brilliant Blue or silver staining.
14. The remaining sample is spiked on a sequencing filter membrane and used for protein sequence analysis.
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