Purification experiments of non-muscle actin
Purification experiments of non-muscle actin
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Purification of non-muscle actin
Materials and Instruments
Non-muscle actin Move 1. Prepare storage solution and materials. For more product details, please visit Aladdin Scientific website.
DNA Enzyme I Buffer
DEAE column
2x DNAzyme I buffer:
20 mmol/L Tris-HCl, pH 8.0
1 mmol/L DTT
0.4 mmol/L CaCI2
0.4 mmol/L ATP
Protease inhibiting peptide
Protease inhibitor
Leupeptin inhibitor peptide
Gastric enzyme inhibitor peptide A
PMSF
TPCK
50% formaldehyde in DNA enzyme column buffer
0.4 mol/L NH4CI in DNAzyme I buffer
0.3 mol/L KCl in DNAase I buffer
DNAase I column buffer
2. Prepare a 5~10 ml affinity column for DNAase I.
3. Prepare a 0.2 ~ 1 ml DEAE column: DEAE-cellulose equilibrated with DNAase I buffer.
4. Collect the cells by centrifugation and wash off the culture medium with isotonic salt solution (e.g. PBS).
5. Resuspend the cell precipitate in DNAase column buffer with protease inhibitor.
6. Homogenize the cells using standard methods for cells of interest.
7. Confirm that more than 99% of the cells have been lysed by phase contrast microscopy.
8. Centrifuge lysates at 100,000 g for 45 minutes.
9. Transfer the supernatant with a Pasteur pipette to avoid mixing with fat.
10. Sample the column:
(1) Mix supernatant with DNAase I Affinity Medium that has been equilibrated with DNAase I Buffer plus Protease Inhibitor and pour the medium/protein mixture into a small rod.
(2) The effluent is collected as the column is loaded, and a significant amount of actin remains in the column as confirmed by immunoblotting with an anti-actin antibody, DNAase I inhibition analysis, or SDS-PAGE.
11. Wash the column with 2x volume of DNAzyme column buffer containing protease inhibitor followed by DNAzyme column buffer containing protease inhibitor and 0.4 mol/L NH4Cl.
12. Elute actin with 50% formaldehyde in DNAase I column buffer.
13. Immediately upsample the 50% formaldehyde eluate onto a 0.2-1 ml DEAE column.
14. Wash the DEAE column with 5 times the volume of DNAase I column buffer, then elute with DNAase I column buffer containing 0.3 mol/L KCl. Collect in sections of 200 μl per tube.
15. Detect the protein concentration in the collection solution, and then mix the collection solution at the peak.
16. Add MgCl2 to a concentration of 2 mmol/L (this was chosen for natural actin) to polymerize actin, and analyze the peptide composition by SDS-PAGE.
The yield should be about 5%, so it is reasonable to expect 2.5-5 mg of purified actin from 10 g wet weight cells.