rat anti tcp 1α  (Santa Cruz Biotechnology)

Journal:

Article Title: Type D Retrovirus Gag Polyprotein Interacts with the Cytosolic Chaperonin TRiC

doi: 10.1128/JVI.75.6.2526-2534.2001

Figure Lengend Snippet: Coimmunoprecipitation of M-PMV Gag polyprotein with the chaperonin TRiC. Cell fractionation and coimmunoprecipitation were carried out with 293T cells transiently cotransfected with M-PMV proviral DNA pSHRM15 and a Myc-tagged TCP-1γ-expressing plasmid. (A) The cells were lysed in 0.5% NP buffer and then fractionated through a continuous 5-to-40% (wt/vol) linear sucrose gradient. The fractions were collected from bottom to top and measured for sucrose density (top) Aliquots of each fraction were analyzed by Western blot assay with rat monoclonal anti-TCP-1α (TCP-1α), mouse monoclonal anti-Myc (TCP-1γ–Myc), and rabbit anti-Gag (Pr78gag). The densities of fractions 6 and 7 (1.076 and 1.086 g/ml, respectively) are near the expected density of the TRiC complex. (B) Peaks I and II (pooled fractions 6 and 7 and 11 and 12, respectively) were diluted with 0.5% NP buffer and then used to coimmunoprecipitate TCP-1γ–Myc and its associated proteins with a mouse monoclonal anti-Myc antibody. Proteins in the immune complex were analyzed as described above for TCP-1γ–Myc, TCP-1α, and Gag by Western blot assay.

Article Snippet: Proteins in the immune complex were separated by 8%–SDS PAGE and were analyzed by Western blot assay with a mouse monoclonal anti-Myc antibody, a rat monoclonal anti-TCP-1α antibody (StressGen), and a purified rabbit anti-Gag antibody.

Techniques: Cell Fractionation, Expressing, Plasmid Preparation, Western Blot

Journal:

Article Title: Type D Retrovirus Gag Polyprotein Interacts with the Cytosolic Chaperonin TRiC

doi: 10.1128/JVI.75.6.2526-2534.2001

Figure Lengend Snippet: Effects of ATP and ATP-γ-S on the M-PMV Gag association with the chaperonin TRiC. 293T cells transiently cotransfected with an M-PMV proviral DNA and a TCP-1γ–Myc-expressing plasmid were lysed in 1% NP-40-containing TNE buffer. (A) Cell lysates were incubated with 2 mM MgCl2 and various concentrations of ATP on ice for 1 h prior to coimmunoprecipitation with mouse monoclonal anti-Myc antibody. TCP-1γ–Myc and Gag proteins were detected by Western blot assay with mouse anti-Myc and rabbit anti-Gag antibodies, respectively. The sample with no ATP was sham treated with H2O. (B) To confirm the effect of ATP on dissociation of Gag proteins from TRiC, competition assays were carried out with nonhydrolyzable ATP analog ATP-γ-S. Cell lysates were treated for 1 h on ice with various concentrations of ATP-γ-S in the presence of 0.1 mM ATP and 2 mM MgCl2. After coimmunoprecipitation with an anti-Myc antibody, TCP-1γ–Myc, TCP-1α, and Gag proteins were detected as described above with mouse anti-Myc, rat anti-TCP-1α, and rabbit anti-Gag antibodies, respectively.

Article Snippet: Proteins in the immune complex were separated by 8%–SDS PAGE and were analyzed by Western blot assay with a mouse monoclonal anti-Myc antibody, a rat monoclonal anti-TCP-1α antibody (StressGen), and a purified rabbit anti-Gag antibody.

Techniques: Expressing, Plasmid Preparation, Incubation, Western Blot