Goat anti rat igg hrp

Fly head samples were prepared in a lysis buffer solution (50 mM Tris-buffered saline (Tris-HCL) pH 7.5, 150 mM NaCl, 1% Triton X100) with protease inhibitor cocktail (Thermo Scientific, #87786; 1:100). Samples were centrifuged for 15 min at 13,300 rpm and the supernatants were collected into new tubes. Subsequently, their protein amount was measured using Bradford protein assay. Quantified proteins were mixed with a solution containing 9:1 ratio of Laemmli buffer (Bio-Rad, #161–0747) to 2-mercaptoethanol (BIOSESANG, #60-24-2) and were boiled at 95°C for 5 min. Samples were then loaded onto Mini-PROTEAN TGX Stain-Free, 4–15% gel (#BR456-8083, Bio-Rad). Protein transfer to the membrane (PVDF) was followed by an incubation in 5% skim milk diluted in 1% TBST (blocking buffer) for 1 hr at RT. Then they were incubated with primary antibodies overnight at 4°C. The follow primary antibodies were used: Rabbit anti-TBPH (from Dr. C.-K. James Shen) (1:5000), Rat anti-elav (DSHB 7E8A10) (1:200,000). After washing, the membranes were then incubated for 1 hr at RT with the corresponding secondary antibodies: Goat anti-rat IgG-HRP (Santa Cruz, sc-2006) (1:100,000) and Goat anti-rabbit IgG HRP (Santa Cruz, sc-3837) (1:5000). Finally, after washing five times in 1% TBST at RT, the membranes were incubated with ECL solution prior to detection using ChemiDocTM XRS+.

Western blot was performed according to the method reported by Arata et al.
[31 (link)]. Briefly, samples including crude somatic extracts of eggs, L3, xL3, L4 and adult worms were electrophoresed on a 12% SDS-PAGE gel. Then, the proteins were electro-transferred onto a nitrocellulose membrane. After being blocked with 5% (w/v) skimmed milk powder in PBST (PBS with 0.5% Tween-20) overnight at 4°C, the membranes were incubated with the primary antibody against rHco-gal-m (dilutions 1:50) for 1 h at 37°C. The membranes were washed 15 min × 3 with PBST and then incubated with the secondary antibody goat anti-rat IgG-HRP (Santa Cruz, USA) in PBST for another 1 h at 37°C. After three washes, the immunoreaction was visualized using ECLsystem (Amersham Biosciences, UK).

A rabbit polyclonal antibody that binds a C-terminal peptide of ovastacin^395-408 and monoclonal antibody M2c.2 that binds to the C-terminal region of ZP2 have been characterized previously [32 (link)][45 (link)]. The following antibodies and lectins were obtained commercially: LCA-FITC (Sigma-Aldrich); antibodies to GP73, calregulin and EEA1, Alex Fluor 488 goat anti-rabbit IgG (H+L)(Life Technologies-Invitrogen, Carlsbad, CA); Alexa Fluor 555 donkey anti-rabbit IgG (H+L) (Life Technologies-Invitrogen); DyLight 649 goat anti–rabbit IgG (H+L) (Life Technologies-Invitrogen); and goat anti-rat IgG-HRP (Santa Cruz).

Protein lysate preparation and western blotting were performed as previously described with the following change [62 (link)]. PVDF membranes were used and membranes were pre-wet in methanol for two minutes and then incubated in transfer buffer for five minutes. The following primary antibodies were used: rat anti-HSF1 mAb (Enzo Lifesciences, Farmingdale, NY), rabbit anti-HSP27 pAb (Enzo), rabbit anti-DNAJB1/HSP40β pAb (Enzo), rabbit anti-DNAJC17/HSP40C pAb (Abcam, Cambridge, United Kingdom), mouse anti-HSP70/72 mAb (Enzo), rat anti-HSP90α mAb (Enzo), mouse anti-HSP90β mAb, rabbit anti-HSP105/110 pAb (Enzo), rabbit anti-HSF1 phospho-serine (pS) 326 (Abcam), and rabbit anti-HSF1 pS303 (Abcam). The following secondary antibodies were used: ECL Rabbit IgG HRP-linked whole Ab (from donkey) (GE Healthcare), ECL Mouse IgG HRP-linked fragment Ab (from sheep) (GE Healthcare) [for all mouse antibodies except anti-HSP90β], goat anti-mouse IgG HRP (PerkinElmer Life Sciences, Boston, MA) [for anti-HSP90β], and goat anti-rat IgG HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Western blot analysis was performed as described previously [40 (link)]. Protein lysates were prepared by lysing cells in trichloroacetic acid. Proteins were analyzed by SDS–polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. In addition, Ponceau S staining of the membrane was performed (loading control). Subsequently, the membrane was hybridized with either rat anti-HA High-Affinity antibody (Roche; 3F10; detects SHIP1 only after lentiviral transduction), mouse anti-SHIP1 antibody (P1C1; Santa Cruz; detects endogenous SHIP1, including after lentiviral transduction), mouse anti-HSC70 antibody (B-6) (both Santa Cruz), rabbit anti-MAPK antibody (9212; Cell Signaling), rabbit anti-phospho-AKT antibody (9018; Cell Signaling), rabbit anti-pan-AKT (11E7; Cell Signaling), rabbit anti-phospho-GSK3β-S9 (9336; Cell Signaling), rabbit anti-phospho-S6-ribosomal protein S240/244 (2215; Cell Signaling) and mouse anti-GAPDH antibody (32233; Santa Cruz). Further antibodies used were anti-mouse IgG HRP-conjugated, anti-rabbit IgG HRP-conjugated (both Cell Signaling) and goat anti-rat IgG HRP (Santa Cruz). Subsequently, protein expression was quantified using LAS-3000 Imager from Fuji (Raytest).