Belgium (English)
-
Your selected country is
Belgium
- Change country/language
nand
Products
Solutions
Discover & Learn
Resources & Tools
Support
You are now leaving the BD Biosciences website. The site you are about to visit is operated by a third party. The link to this site neither makes nor implies any representation or warranty for any products or services offered on a third-party site and is intended only to enable convenient access to the third-party site and for no other purpose. Do you want to continue?
Old Browser
For the best web browsing experience, please use Chrome, Safari or Firefox, minimum versions 77.0.3865, 12.1.2 and 68, respectively.
Immunopurification of Tyrosine Phosphorylated Proteins
Cell Lysis
Non-Denaturing Conditions
- Rinse adherent or non-adherent cells from a 15 cm dish with an excess of phosphate-buffered saline (PBS); repeat wash.
- Lyse cells with 6 ml of ice-cold lysis buffer (10 mM imidazole pH 7.3, 0.5 M NaCl, 1% Triton X-100, 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 2 mM sodium azide) with gentle rotation for 30 minutes at 4°C.
- Remove the lysate from the plate and centrifuge at 40,000 rpm for 1.5 hour at 4°C.
Denaturing Conditions
- Rinse adherent or non-adherent cells from a 15 cm dish with an excess of phosphate-buffered saline (PBS); repeat wash.
- Lyse cells with 3 ml of boiling lysis buffer (1% SDS, 10 mM Tris pH 7.4, 0.2 mM sodium ortho-vanadate).
- Microwave the cells for 5 seconds to assure complete lysis and denaturation.
- Scrape the lysate from plates and centrifuge at 40,000 rpm for 1.5 hour at 15°C.
- Dilute the lysate 5-fold to a final concentration of 0.2% SDS, adjusted to 10 mM imidazole pH 7.3, 50 mM NaCl, 1% Triton X-100, 0.2 mM sodium ortho-vanadate, 1 mM EDTA.
Purification
- Equilibrate PY20 coupled Affi-Gel 10 (Bio-Rad) in cold resin wash buffer (10 mM imidazole pH 7.3, 50 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA) by repeated centrifugation and resuspension in resin wash buffer.
- Incubate the lysate (up to 250 mg total protein) with 3 ml of anti-phosphotyrosine PY20 coupled to Affi-Gel 10 overnight at 4°C with constant rotation.
- Collect the Affi-Gel by low speed centrifugation, resuspend in resin wash buffer, and load into a small column.
- Wash the column with 20 volumes of resin wash buffer.
- Wash the column with three volumes of low-detergent resin wash buffer (10 mM imidazole pH 7.3, 50 mM NaCl, 0.1% Triton X-100, 0.05% SDS, 1 mM EDTA).
- Elute the tyrosine-phosphorylated proteins with 5 mM phenyl phosphate in the low-detergent buffer. The eluted proteins can be monitored by protein determination or Western blotting with anti-phosphotyrosine antibodies.
- The column should be regenerated by extensive washing with a neutral pH buffer (like PBS) containing 1 M NaCl, followed by several washes with PBS alone. Store the column at 4°C in PBS + 1.5 mM sodium azide between uses.
References
- Glenney, Jr., J.R. and Zokas, L. 1989 J. Cell Biol. 108: 2401
- Glenney, Jr., J.R. 1991 Meth. Enzymology 201: 92
APO-BRDU and APO-DIRECT are trademarks of Phoenix Flow Systems.
23-23971-00
Report a Site Issue
This form is intended to help us improve our website experience. For other support, please visit our Contact Us page.
Successfully submitted.