mouse anti v5 monoclonal primary antibody  (Bio-Rad)

Journal: Nature Communications

Article Title: Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells

doi: 10.1038/ncomms6339

Figure Lengend Snippet: ( a ) Kinesin-5 and kinesin-14 constructs used in Fast Protein Liquid Chromatography. V5-tagged Cut7 and two truncation constructs were used, in addition to one FLAG-Pkl1 truncated construct that retains full Pkl1 activity. Cut7 constructs are V5-tagged full-length Cut7 (aa 1–1,085), Cut7-Head-Stalk (Cut7HS, aa 1–888) and Cut7-Stalk-Tail (Cut7-ST, aa 443–1,085). ( b ) Western blot profiles of whole-cell extracts fractionated by Separose 6 using FPLC. ( c ) Western blots of Cut7 constructs immunoprecipitated from whole-cell extracts using anti-V5 magnetic beads with empty strain negative controls. ( d ) Cartoon diagram of 6-His tagged Pkl1 Tail peptide co-immunoprecipitation assay using magnetic beads with His affinity and FPLC fraction 15. ( e ) Pkl1 Tail peptide co-immunoprecipitation of γ-TuRC core subunits and V5-Cut7ST using a short Pkl1 Tail peptide (PγT). Mutated peptide PγM has significantly reduced interaction with the fission yeast γ-TuRC. The anti-HA antibody detects the HA-tagged γ-TuRC protein Alp4.

Article Snippet: Antibodies used were primary mouse anti-γ-tubulin monoclonal (1:10,000; Sigma-Aldrich cat. T5326), primary rabbit anti-HA epitope tag (1:5,000; Rockland cat. 600-401-384), primary rabbit anti-FLAG polyclonal (1:320; Sigma-Aldrich cat. F7425), primary mouse anti-V5 monoclonal (1:5,000; Life Technologies cat. R96025) or mouse anti-V5 IgG HRP conjugated monoclonal (1:5,000; Life Technologies cat. R96125), goat anti-rabbit IgG HRP conjugate (1:20,000; Millipore cat. 12-348) and goat anti-mouse IgG HRP conjugate (1:10,000; Novagen cat. 71045).

Techniques: Construct, Fast Protein Liquid Chromatography, Activity Assay, Western Blot, Immunoprecipitation, Magnetic Beads, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells

doi: 10.1038/ncomms6339

Figure Lengend Snippet: ( a ) FPLC profiles of V5-tagged Cut7 and two truncation constructs in γ-tubulin helix 11 mutant gtb1-K5A . ( b ) Structural model of γ-tubulin-K5A and -PL302 mutants (right) shown with respect to the α-/β-tubulin heterodimer (left). β-tubulin helix 11 is a conserved docking site for Klp Motor domains, and is additionally conserved with fission yeast γ-tubulin helix 11. ( c ) Fluorescence localization and steady-state expression levels from whole-cell extract of full-length V5-Cut7 in wild-type gtb1 versus the gtb1-K5A mutant. ( d ) Fluorescence localization of V5-NLS-Cut7ST (Cut7ST, aa 443–1,085) and V5-Cut7HS in the gtb1-K5A strain. ( e ) Fluorescence localization of four cut7 deletion and BimC site-directed mutagenesis derivatives generated in this study in pkl1Δ cut7Δ cells fixed at 36 °C. Deletion constructs used are V5-tagged NLS-Cut7-Stalk-Tail, NLS-Cut7-Stalk-Tail (Cut7ST , Pro to Ser at aa 1,021), NLS-Cut7-Tail (Cut7T, aa 888–1,085) and NLS-Cut7-Tail (Cut7T , Pro to Ser at aa 1,021). Scale bars, 5 μm.

Article Snippet: Antibodies used were primary mouse anti-γ-tubulin monoclonal (1:10,000; Sigma-Aldrich cat. T5326), primary rabbit anti-HA epitope tag (1:5,000; Rockland cat. 600-401-384), primary rabbit anti-FLAG polyclonal (1:320; Sigma-Aldrich cat. F7425), primary mouse anti-V5 monoclonal (1:5,000; Life Technologies cat. R96025) or mouse anti-V5 IgG HRP conjugated monoclonal (1:5,000; Life Technologies cat. R96125), goat anti-rabbit IgG HRP conjugate (1:20,000; Millipore cat. 12-348) and goat anti-mouse IgG HRP conjugate (1:10,000; Novagen cat. 71045).

Techniques: Construct, Mutagenesis, Fluorescence, Expressing, Generated