Donkey anti rabbit hrp antibody

Tpm isoform‐specific Tpm antibodies are described by Schevzov et al. 63: γ9d (sheep polyclonal and mouse monoclonal antibodies) recognizes the 9d exon from the Tpm3 gene (both human and mouse) corresponding to Tpm3.1 (Tm5NM1) and Tpm3.2 (Tm5NM2); CG3 (mouse monoclonal antibody) recognizes the 1b exon from the Tpm3 gene, which is contained in all cytoskeletal (non‐muscle) Tpms 64. Primary antibodies used: Akt and phospho‐Akt (Ser473) rabbit polyclonals (1:1000; Cell Signalling Technology, Inc.); GLUT4 (1F8) mouse monoclonal (1:3000 for western blots; 1:200 for immunofluorescence staining) (from David James) 65; rSec8 mouse monoclonal (Stressgen); Myo1c (M2) mouse monoclonal 66; syntaxin 4 rabbit polyclonal (Synaptic Systems); α‐tubulin mouse monoclonal (DM1a) (Sigma) and MyoIIA rabbit polyclonal (Covance). Secondary antibodies used for western blot analysis: anti‐rabbit, anti‐sheep and anti‐mouse IgG‐conjugated horseradish peroxidise (HRP) (GE Healthcare) and donkey/anti‐rabbit/HRP antibodies (for Akt western blots) (Jackson ImmunoResearch Laboratories).
The anti‐Tpm compound TR100 has been designed to target Tpm3.1 using the sequence divergence at the C‐terminus of cytoskeletal versus muscle Tpms. This compound targets Tpm3.1 and disrupts actin filaments by inhibiting the actin filament‐stabilizing action of Tpm3.1 41.

Wells coated with purified complement components C3, C4, C5 WT or C5 R885H were used to assess the binding of our nanobodies and Ecu-mab to different complement proteins. Nanobodies and Ecu-mab were added in a 10-fold titration (0.001–1000 nM). Next, wells containing nanobodies were incubated with 1:2000 primary polyclonal rabbit-anti-VHH antibody QE19 (QVQ Holding BV) and 1:5000 secondary polyclonal donkey-anti-rabbit-HRP antibodies (Jackson ImmunoResearch). To detect Ecu-mab binding, wells were incubated with anti-human-kappa-HRP (Southern Biotech) detection antibodies.

Wells coated with C5 were incubated with llama serum to measure the presence of C5-binding llama antibodies. Llama serum was diluted in PBS, supplemented with 1% skimmed milk (Marvel). Llama antibodies were detected using 1:2000 primary polyclonal rabbit-anti-VHH antibody QE19 (QVQ Holding BV) and 1:5000 secondary polyclonal donkey-anti-rabbit-HRP antibodies (Jackson ImmunoResearch). ELISA was developed using 3.7 mM O-phenylenediamine dihydrochloride + 50 mM Na₂HPO₄ 2H₂O + 25 mM citric acid + 0.03% H₂O₂ and the reaction was stopped with 0.5 M sulfuric acid.