1. Prepare buffers and have them cold. Make 50X stock of Buffer G and dilute as needed from frozen aliquots of 50X Buffer G.
1X Extraction Buffer without detergents: 1 M Tris-Cl, pH 7.0, 0.6 M KCl, 0.5 mM ATP, 1 mM DTT, 0.5 mM MgCl2, 0.2 mM PMSF (add just before use). Buffer D: 10 mM Tris-Cl, pH 8.0, 0.2 mM CaCl2 , 0.5 mM ATP, 0.1 mM DTT, 100 mM KCl, 0.1 mM PMSF. Buffer G: 1 mM Tris-Cl, pH 8.0, 0.2 mM ATP, 0.2 mM CaCl2 , 0.1 mM DTT, 0.005% NaN3
2. Make sure there is alot of distilled water in the cold room carboys.
3. Prepare 1.5 liters of DE-53 cellulose and equilibrate in Buffer D. Keep the resin in a 4 l beaker and saturate with ATP by adding 7.5 g ATP, stir 30 min in the cold and wash resin 3 times with Buffer D (ATP saturation described further in Ref 3 ).
4. Acquire a Polytron or other homogenizer.
5. Have DNaseI-Sepharose column equilibrated in Buffer G and hooked in tandem with a G-25 Sephadex column also equilibrated in Buffer G. Directions for preparation of the DNaseI column are below in Appendix .
Day 1 Extraction of Actin from Chicken Brain (large scale prep from tissue)
1. Homogenize tissue (fresh or frozen) in 1X Extraction Buffer without detergents at a ratio of 100ml buffer for 10 g tissue using a Polytron homogenizer (large chopper). If using frozen tissue, thaw the tissue in buffer by swirling briefly in buffer in a 37 °C water bath before homogenizing. Run homogenizer in 3-4 10 second bursts with the beaker on ice. Add Triton X-100 and Tween-20 to the homogenate at final concentrations of 4% (v/v) and 1 mg/ml, respectively, and homogenize briefly to mix. Stir the homogenate on ice for 30 min.
2. Centrifuge at 25,400 x g for 30 min (Sorvall GSA rotor, 12,500 rpm). Collect supernatant and centrifuge at 100,000 x g for 1 hour (Ti 45, 40,000 rpm). Filter the supernatant fraction through cheesecloth to remove lipid.
3. Dialyze the filtered supe against 10 l of cold water for 4 hours, followed by dialysis overnight with two changes of Buffer D. Any precipitate that formed during dialysis is removed the next day by centrifugation at 25,400 x g for 30 min (Sorval GSA rotor, 12,500 rpm).
Day 2 DE-53 cellulose chromatography
1. Drain the excess Buffer D from the DE-53 cellulose (1.5 l) that was equilibrated in Buffer D and saturated with ATP. Add the tissue extract and stir the resin for 30 min using a large stir plate in the cold room. Transfer the resin to a column (10 cm in diameter) and wash the column with 3 l of Buffer D using a fast flow rate.
2. Elute the column with a 4 l gradient from 100 mM KCl to 500 mM KCl in Buffer D. Collect 25 ml fractions (400 drops/fraction). Actin-containing fractions are identified by dot blot using mAb C4 (see below) and pooled.
3. Dot blots to identify actin in the DE-53 column fractions are performed by spotting 2 ml of each fraction tested to a sheet of nitrocellulose marked with a grid pattern in pencil. After the samples were air dried, the nitrocellulose is soaked for 15 min in 0.039 M glycine, 0.048 M Tris base, 0.0375% SDS and 20% methanol. Rinse the blot in TTBS (0.3 M NaCl, 20 mM Tris/Cl, pH 7.8, 0.1% (v/v) Tween-20 and 0.01% NaN 3 ), block in gelatin/TTBS and react the blot with mAb) C4 followed by reaction with alkaline phosphatase-conjugated goat anti-mouse secondary antibody. Choose the actin pool broadly, however, consider also checking the column fractions by dot blot with mAB 3F2 to identify capping protein. Pool the actin fractions but do not take fractions rich in capping protein, which elutes before actin in the gradient.
Day 3 Affinity chromatography on DNase I-Sepharose.
1. Have DNaseI-Sepharose (5ml volume) packed into a 2.5 cm diameter column and equilibrated in Buffer G. Have the tubing and connections ready to connect the column in tandem with a G25 column (2.5 x 44 cm), also equilibrated in Buffer G, but don''t have them connected yet. The tubing between the outflow of the DNaseI column and the inlet of the G25 column should be as short as possible.
2. Apply the DE-53 pool to the Sepharose-DNAse I column (not yet connected to the G25 column). Collect flow-through and save it.
3. Wash column with Buffer G until the A280 of the eluate is <0.010. Then wash with 60 ml of Buffer G containing 10% formamide. Next, wash the column with 60 ml Buffer G containing 10% formamide and 0.2 M NH4 Cl. Finally, wash the column with 60 ml of Buffer G. Collect eluates in bulk for all these washes.
4. Connect the outlet of the DNaseI column to the inlet of G25 column. Elute actin from the DNaseI column with 60 ml G-buffer containing 50% formamide (this buffer must be ice cold to minimize denaturation of the actin). Collect 20-25 fractions (64 drops/fraction).
5. Disconnect the DNaseI column and continue to elute the G25 column with Buffer G. Monitor the A290 of the fractions to find the peak of protein, which is all actin.
6. If desired, the actin obtained from the DNase I-Sepharose column can be concentrated by absorption to a 1 ml aliquot of DE-53 cellulose equilibrated in Buffer D and saturated with ATP as described above. After absorption, the resin is placed in a column and the actin is eluted with Buffer D containing 0.4 M NaCl. Alternatively, chromatography on a MonoQ column with elution in a steep gradient from 0.1 M NaCl to 0.4 M NaCl in Buffer D can be used in place of the batchwise DE-53 cellulose step.
The actin also can be concentrated by polymerization induced by addition of 1/9 volume of 10X MKEI buffer (1 M KCl, 20 mM MgCl2, 10 mM EGTA and 0.2 M imidazole-HCl, pH 7.0) and incubated overnight at room temperature. Actin was pelleted by centrifugation at 132,00 x g in a Ti75 rotor (Beckman, Palo Alto, CA). The pellet was resuspended using a small Dounce homogenizer in a minimal volume of G-buffer (2 mM TrisCl, pH 8.0, 0.2 mM ATP, 0.1 mM DTT, 0.2 mM CaCl2 , 0.005% NaN3 ) and dialyzed against several changes of G-buffer. The sample was clarified by centrifugation at 70,000 rpm in a TLA-100.3 rotor (Beckman, Palo Alto, CA), and the upper 3/4 of the supernatant was collected. The actin was stored on ice or flash frozen in aliquots using liquid N2 .
Modification for isolation of Erythrocyte Actin
1. Collect blood from animal using 20 ml/100 ml whole blood acid citrate/dextrose (0.085 M sodium citrate, 0.065 M citric acid 2% dextrose) as anti-coagulate.
2. Centrifuge blood at for 10 min at 4300 x g and remove the serum and buffy coat. Wash the erythrocytes three times in Tris-buffered saline (10 mM Tris-Cl, pH 7.4, 146 mM NaCl and 0.01 PMSF).
3. Lyse the packed cells 5 by addition of an equal volume of 2X extraction buffer (2 M Tris-Cl, pH 7.0, 1.2 M KCl, 1 mM ATP, 2 mM DTT, 1 mM MgCl2 , 2 mg/ml Tween-20, 7.5% (w/v) Triton X-100 and 0.2 mM PMSF) and stir gently at 30°C for 30 min. The extract was dialyzed against Buffer D (10 mM Tris-Cl, pH 8.0, 0.2 mM CaCl2 , 0.5 mM ATP, 0.1 mM dithiothreitol (DTT), 100 mM KCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)) and applied batchwise to approximately 1.5 l of DE-53 cellulose as described above. After packing the resin into the column, wash the resin until the eluate is no longer red in color.
Appendix: How to prepare the DNaseI column.
Variant 1. Coupling DNase I to CNBr-Sepharose
1.Deoxyribonuclease I (Worthington biochemical corporation, Halls Mill Rd. Freehold, New Jersey.07728) 54B237, 100 mg, $211.
Dissolved in 10 ml of 0.1 M Na Borate pH 9, 0.1mM CaCl2 , 1 mM PMSF. Dialysed vs 1 L of the same buffer w/o PMSF. OD280 measured after dialysis.
Coupling Buffer : 0.1M NaHCO2 , pH 8.5, 0.5M NaCl.
For 1 liter: 8.4g NaHCO2 , 29.2g NaCl, pH to 8.5 with 50% NaOH.
2. CNBr-Sepharose resin must be "activated" just before use so don''t procede unless everything else is ready. The "activated" resin is not stable for long so read this protocol in advance so that things can proceed smoothly . Cyanogen bromide activated sepharose 4B (Sigma) C-9142 5g. This amount swells to 20 ml volume . Swell resin in 10 mM HCl (86 ml of conc. HCl into 100 ml H2 O) for 15 min at room temperature. Have a scintered glass filter ready to wash resin. Pour the swelled resin into the filter and gently wash under a vacuum with 100 ml 10 mM HCl. Wash with coupling buffer and then transfer the resin quickly to a solution of protein .
3. Incubate resin and protein overnight on a rotator at 4°C (cold room).
4. Gently centrifuge the resin (low g''s) and collect the supernatant and SAVE IT . Measure the A280 of the supernatant and determine that the coupling worked. The A280 of this supernatant should be way less than that of the starting protein solution. A good coupling will give an ending A280 of ~10 fold less than the starting A280 .
5. Transfer the resin to a small column (I use 5ml dispo-columns) and wash the resin with sequential 10 ml aliquots of coupling buffer and wash buffer . Do the sequential washes at least 5 times, ending with an extensive wash (25ml) in TTBS, containing an extra 0.2 M NaCl. The column is now ready for use. For long term storage, cap bottom of column and wrap Parafilm around the top and store at 4°C (there is azide in the TTBS).
Wash Buffer : 0.1M Na Acetate, pH 4.0, 0.5M NaCl.
For 1 liter: 5.25ml glacial acetic acid, 29.2g NaCl, check pH to get 4.0 and adjust with 50% NaOH if necessary.
TTBS : 0.3M NaCl, 20mM TrisCl, pH 8.0, 0.05% Tween-20 [note: I add extra NaCl to TTBS to give a final concentration of 0.5M NaCl, i.e., add 25ml of 4M NaCl to 500ml of our normal TTBS].
Variant 2. Coupling DNase 1 to Affigel 10
1. Wash one bottle of Affigel 10 Gel (BioRad) into a Buchner funnel with a sintered glass filter. Wash well with cold water to remove the isopropanol but do not allow the gel to dry.
2. Transfer the cake of gel into a 50 ml screw-cap tube. Add Hepes, pH 7.4 and Ca Cl2 to make a final concentration (including gel volume) of 0.1 M and 2 mM respectively.
3. Add 100 mg of DNase 1 (Boehringer - Mannheim) Grade II, $37. Shake gently at 4° for 4-5 hr.
4. Spin down the gel at 500xg for 5 min. Remove the supernatant and add 0.5M Tris pH 7.5, to block any remaining active sites. Shake gently 4-5 hr at 4°.
5. Spin down the gel at 500xg for 5 min. Remove the supernatant and wash the gel with 3 M guanidine-HCl containing 0.5 M sodium acetate. Load it into the column.
6. Remove the guanidine solution and wash the gel well with 10mM tris pH 7.5, 2 mM CaCl2 . Store the column in this solution containing 0.02% NaN3 .
1. Pinder, et al., (1995) Concentrated Tris solutions for preparation, depolymerization and assay of actin: application to erythroid actin. Anal. Biochem. 225:291-295.
2. Kron, et al., (1992) PNAS 89:4466-4470 -- DNaseI affinity chromatography.
3. Gordon, et al., (1976) JBC 251:4778-4786 -- DEAE cellulose chromatography of actin