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Protocol used by Liang in Bear Lab - UNC at Chapel Hill
Polyclonal antibody affinity purification
The protocol is specific for Coronin 1B polyclonal antibody affinity purification, and the purified antibody was named as 4245.Exp . Rabbits were immu- nized with a GST fusion protein containing the human Coronin 1B C-terminal region (394-489 amino acids) by Covance. Serum was affinity purified against maltose- binding fusion protein, containing the equivalent region of mouse Coronin 1B to ensure cross-reactivity. This maltose-binding fusion protein was purified in a buffer containing 10 mM MOPS, pH 7.5, and 60 mM sodium citrate, which was used for direct coupling to UltraLink biosupport media (Pierce) according to the manufac- turer’s protocol. The column was washed extensively with 10 mM Tris pH 7.5, 100 mM glycine pH 2.5, and 100 mM triethylamine pH 11.5, with neutralizing washes between. Crude serum was diluted 10-fold in 10 mM Tris, pH 7.5, and passed through the column three times. After loading the antigen, the column was washed with 10 mM Tris pH 7.5, and then with 500 mM NaCl, 10 mM Tris pH 7.5. Antibodies were eluted sequentially with 100 mM glycine pH 2.5, followed by 100 mM triethy- lamine pH 11.5. The antibody-containing fractions were neutralized, combined, and dialyzed against phosphate-buffered saline. Below is a step-by-step protocol: 1. Prepare MBP-m1Btail purification buffers. Base Buffer: 0.01 M MOPS pH 7.5,
0.06 M sodium citrate. Elute Buffer: 0.1 M MOPS pH 7.5, 0.6 M sodium citrate,
2. Start a 50 ml culture with Ampicillin to 1× (50 µl of 1000× stock). Culture at 3. Add 50 ml starter culture to 1 liter LB Media. Add Ampicillin.
Protocol used by Liang in Bear Lab - UNC at Chapel Hill
5. Cool culture on ice to room temperature. Add IPTG to 0.1 mM.
6. Shake at room temperature for ∼6 hours.
7. Spin down cells (500 ml bottles, GS-3 rotor, 4700 rpm, 15 min). Pour off media.
Keep pellets in -20◦C or directly proceed the next step.
8. Warm cell pellet on ice. Resuspend it in Base Buffer (1 liter culture to 50 ml).
Sonicate for 6 minutes on ice(15-sec on, 15-sec off, 10 ml each sonication).
9. Freeze sample in a dry ice ethanol bath. Thaw in cold water. Rotate at cold 10. Spin the lysate at 27000×g (JA17 rotor, 14000 rpm, 15 min).
11. Dilute the supernatant 1:3 with Base Buffer. Apply it to Base Buffer equili- brated amylose resin column (New England Biolabs, 1 ml 50% slurry for 330 12. Wash the column with 15 ml Base Buffer. Elute the column with 2×3 ml Elute 13. Regenerate the amylose resin column with 3 ml water; 3 ml 0.1% SDS; 3 ml 14. Coupling the antigen to a bead support (UltraLink from Pierce etc.). In most circumstances, efficient coupling should exceed 90%. Extensive coupling may lead to the loss of the antibody binding sites. 15. Transfer the beads with the bound antigen to a column. Wash with 10 bed- volumes of 10 mM Tris (pH 7.5). Then wash with 10 bed-volume of 100 mM glycine (pH 2.5), followed by 10 mM Tris (pH 8.8) until the pH of last drop Protocol used by Liang in Bear Lab - UNC at Chapel Hill
to be 8.8. Then 10 bed-volumes of 100 mM triethylamine (pH 11.5, prepared fresh). Wash with 10 mM Tris (pH 7.5) until the pH reaches 7.5.
16. Clean the serum by centrifugation. Dilute the whole serum 10-fold by 10 mM Tris (pH 7.5). Pass the serum through the column for three times.
17. Wash the column with 20 bed-volumes of 10 mM Tris (pH 7.5), and then with 20 bed-volumes of 500 mM NaCl, 10 mM Tris (pH 7.5).
18. Elute with 10 bed-volumes of 100 mM glycine (pH 2.5). Collect each bed- volume elute in a tube containing 1/10 bed volume of 1 M Tris (pH 8.0).
19. Wash the column with 10 mM Tris (pH 8.8) until the pH rises to 8.8.
20. Elute with 10 bed-volumes of 100 mM triethylamine (pH 11.5, prepared fresh).
Collect each bed-volume elution in a different tube containing 0.1 bed-volume 21. Wash the column with 10 mM Tris (pH 7.5) until the pH is 7.5. The column may be re-used by storing it in buffers with 0.01% merthiolate.
22. Blot 1 µl of the elutions onto the membrane. Use the appropriate secondary 23. Combine the antibody fractions and dialyze against PBS (add 0.02% sodium azide for store). If necessary, concentrate it by ammonium sulfate precipitation Bibliography
Bear, J. E., Loureiro, J. J., Libova, I., Fassler, R., Wehland, J., and Gertler, F. B. (2000).
Negative regulation of fibroblast motility by Ena/VASP proteins. Cell, 101(7):717–728.
Bear, J. E., Svitkina, T. M., Krause, M., Schafer, D. A., Loureiro, J. J., Strasser, G. A., Maly, I. V., Chaga, O. Y., Cooper, J. A., Borisy, G. G., and Gertler, F. B. (2002).
Antagonism between Ena/VASP proteins and actin filament capping regulatesfibroblast motility. Cell, 109(4):509–521.
Benting, J., Lecat, S., Zacchetti, D., and Simons, K. (2000). Protein expression in Drosophila Schneider cells. Anal Biochem, 278(1):59–68.
Bordier, C. (1981). Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem, 256(4):1604–1607.
Cai, L., Holoweckyj, N., Schaller, M. D., and Bear, J. E. (2005). Phosphorylation of coronin 1B by protein kinase C regulates interaction with Arp2/3 and cell motility.
J Biol Chem, 280(36):31913–31923.
Cai, L., Makhov, A. M., and Bear, J. E. (2007a). F-actin binding is essential for coronin 1b function in vivo. J Cell Sci, 120(Pt 10):1779–1790.
Cai, L., Marshall, T. W., Uetrecht, A. C., Schafer, D. A., and Bear, J. E. (2007b). Coronin 1b coordinates arp2/3 complex and cofilin activities at the leading edge. Cell,128(5):915–929.
Clainche, C. L. and Carlier, M.-F. (2004). Actin-based motility assay. Current Protocols in Cell Biology, 12:12.7.1–12.7.20.
Dayel, M. J. and Mullins, R. D. (2004). Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysison Arp2. PLoS Biol, 2(4):E91.
Higgs, H. N., Blanchoin, L., and Pollard, T. D. (1999). Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actinpolymerization. Copyright by Liang Cai and James Bear Biochemistry, 38(46):15212–15222.
Protocol used by Liang in Bear Lab - UNC at Chapel Hill
Ichetovkin, I., Grant, W., and Condeelis, J. (2002). Cofilin produces newly poly- merized actin filaments that are preferred for dendritic nucleation by the arp2/3complex. Curr Biol, 12(1):79–84.
Kuhn, J. R. and Pollard, T. D. (2005). Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy. Biophys J,88(2):1387–1402.
Lindroth, M., Bell, P. B., Fredriksson, B. A., and Liu, X. D. (1992). Preservation and visualization of molecular structure in detergent-extracted whole mounts of cul-tured cells. Microsc Res Tech, 22(2):130–150.
Maunsbach, A. B. (1998). Immunolabeling and Staining of Ultrathin Sections in Biological Electron Microscopy. Cell Biology: A Laboratory Handbook, 2nd Edition,3:268–276.
Nagata-Ohashi, K., Ohta, Y., Goto, K., Chiba, S., Mori, R., Nishita, M., Ohashi, K., Kousaka, K., Iwamatsu, A., Niwa, R., Uemura, T., and Mizuno, K. (2004). A path-way of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilinin lamellipodia. J Cell Biol, 165(4):465–471.
Niwa, R., Nagata-Ohashi, K., Takeichi, M., Mizuno, K., and Uemura, T. (2002). Con- trol of actin reorganization by Slingshot, a family of phosphatases that dephos-phorylate ADF/cofilin. Cell, 108(2):233–246.
Ris, H. (1985). The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections. J Cell Biol, 100(5):1474–1487.
Small, J. V. and Sechi, A. (1998). Whole-Mount electron Microscopy of the Cytoskele- ton: Negative Staining Methods. Cell Biology: A Laboratory Handbook, 2nd Edition,3:285–291.
Svitkina, T. M. and Borisy, G. G. (2005). Correlative light and electron microscopy studies of cytoskeletal dynamics, volume 3 of Cell Biology A Laboratory Handbook. ElsevierScience (USA), 3rd edition.
Svitkina, T. M., Verkhovsky, A. B., and Borisy, G. G. (1995). Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells. J StructBiol, 115(3):290–303.
Tosatti, S., Paul, S. M. D., Askendal, A., VandeVondele, S., Hubbell, J. A., Tengvall, P., and Textor, M. (2003). Peptide functionalized poly(l-lysine)-g-poly(ethyleneglycol) on titanium: resistance to protein adsorption in full heparinized humanblood plasma. Biomaterials, 24(27):4949–4958.

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