Detection of in vivo ubiquitinated protein

doi:10.1038/nprot.2007.35

Method Article

Detection of in vivo ubiquitinated protein

https://doi.org/10.1038/nprot.2007.35

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Biochemistry

Computational biology and bioinformatics

ubiquitination

This protocol was used in our Nature Immunology paper

TBS buffer:

50 mM Tris-HCl pH 8.0, 150 mM NaCl

SDS (Sigma) sample buffer (see elsewhere)

(all the buffers need to be supplemented with deubiquitylation enzyme inhibitors ubiquitin aldehyde and NEM at appropriate concentrations)

Triton X-100 (Sigma)

Anti-ubiquitin (clone Ubi-1; Zymed)

Protein G beads

LiCl (Sigma)

DTT (Sigma)

1 Add 50 μl of ice cold TBS to 1×10⁶ B cells.

2 Add 60 μl of TBS containing 2% SDS, mix quickly.

3 Heat denature for 10 min.

4 Place immediately on ice for 5 min.

5 Add 900 μl of TBS containing 1% Triton X-100.

6 Add anti-ubiquitin (clone Ubi-1, 1-3 μg) and incubate at 4 ˚C overnight on rotator.

7 Add Protein G beads (50 μl) to the supernatant , incubate at 4 ˚C for 1 h on rotator.

8 Wash the beads once with 0.5 ml of TBS containing 1% Triton X-100 and 0.1% SDS, and centrifuge (10 000 rpm, 4 ˚C, 1 min).

9 Wash the beads twice with 0.5 ml of TBS containing 1% Triton X-100 and 0.5 M LiCl, and centrifuge (10 000 rpm, 4 ˚C, 1 min).

10 Wash the beads once with 0.5 ml of TBS containing 1% Triton X-100 and centrifuge (10 000 rpm, 4 ˚C, 1 min).

11 Add 50 μl of SDS sample buffer containing 10 mM DTT (Sigma) to the beads, boil at 98 ˚C for 5 min, centrifuge (10 000 rpm, 25 ˚C, 2 min), and apply the supernatant to SDS-polyacrylamide gel. Perform immunoblot.

2 d

Anzelon, A. N., Wu, H. & Rickert, R. C. Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function. Nat Immunol 4, 287-294 (2003).
Sanchez, Y. et al. Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund complete adjuvant alum, and liposomes. Infect Immun 30, 728-733 (1980).
Engel, P. et al. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39-50 (1995).
Gallagher, E. D., Xu, S., Moomaw, C., Slaughter, C. A. & Cobb, M. H. Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation. J Biol Chem 277, 45785-45792 (2002).
Gallagher, E. D., Gutowski, S., Sternweis, P. C. & Cobb, M. H. RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity. J Biol Chem 279, 1872-1877 (2004).
Rush, J. S., Hasbold, J. & Hodgkin, P. D. Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation. J Immunol 168, 2676-2682 (2002).

Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).

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Detection of in vivo ubiquitinated protein

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