Hrp conjugated anti rabbit antibody

Third instar larvae or S2 cells were lysed with lysis buffer and boiled with sample loading buffer at 95 °C for 10 mins [39 (link)]. Prepared samples were loaded on 7.5% SDS-PAGE gel, and protein bands in the gel were transferred to nitrocellulose membrane by wet method. Membranes were blocked with 5% dry milk in TBS-T (140 mM NaCl; 3 mM KCl; 25 mM Tris pH 7.4; 0.1% Tween-20) at room temperature (RT) for 1 h. Membranes were sequentially probed in primary antibody and HRP-conjugated secondary antibody solution diluted with 2% dry milk in TBS-T. After washing, membranes were developed with WESTSAVE-Gold reagent (AbFrontier).
Primary antibodies used are: concentrated anti-Wg (mouse, 1:2000, Developmental Studies Hybridoma Bank (DSHB)), anti-GFP (rabbit, 1:10,000, abcam), anti-αTub (mouse, 1:5000, Sigma-Aldrich), anti-MYC (rabbit, 1:5000, abcam) and anti-Ago (guinea pig, 1:5000, gift from K. H. Moberg). Secondary antibodies used are: HRP-conjugated anti-mouse antibody (1:10,000, Jackson Laboratory), HRP-conjugated anti-rabbit antibody (1:10,000, Jackson Laboratory) and HRP-conjugated anti-guinea pig antibody (1:5000, Jackson Laboratory).

The CCSP protein expression was evaluated in lung homogenates using a Bio-Rad kit. Samples were then adjusted to 5μg/μl in PBS, pH 7.4, and 10μl of each sample were spotted onto a Hybond C membrane (Amersham Pharmacia). The membrane was blocked with 5% fat-free milk in PBS buffer for 1h, before being incubated for 3h with the rabbit primary antibody anti-CC10 1:500 (Santa Cruz Biotechnology) in blocking buffer at room temperature. After washing with TBS–Tween-20 buffer, the membrane was treated with a HRP-conjugated anti-rabbit antibody (Jackson Immunoresearch) and the next handle was as described above for Western blot. http://dx.doi.org/10.17504/protocols.io.bbyhipt6

The total cellular protein was extracted from the resected OS specimens or adjacent normal bone tissue (NT), or cultured cells. Primary antibodies for Western blot were anti-ZNRF2 and anti-α-tubulin (Cell Signaling, San Jose, CA, USA). HRP-conjugated anti-rabbit antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA) was used as the secondary antibody. The ZNRF2 protein levels were first normalized to α-tubulin, and then normalized to experimental control by NIH ImageJ software (Bethesda, MA, USA), as described before [25 (link)].

PF338 tumor cells were seeded into 6-well plates. After a recovery period of 24 h, cells were treated for 72 h with either HDACi (SAHA, valproate), galunisertib or solvent and precipitated with 6% TCA for 1 h, 4°C followed by centrifugation for 10 min at 9000 rpm. The total cellular protein pellets were resuspended in electrophoresis sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5 mM EDTA, 125 mg/ml urea, 100 mM dithiothreitol) to be later loaded on 10% acrylamide gels in equal protein amounts. For immunostaining rabbit anti-E-cadherin (Cell Signaling, 24E10, 1:1,000), anti-beta-catenin (Santa Cruz, Sc-7199, 1:500), anti-pSMAD2 (Cell Signaling, 138D4, 1:1,000) and polyclonal anti-beta-tubulin (Abcam, ab6046, 1:1,000) were used. As secondary antibody HRP-conjugated anti-rabbit antibody (Jackson ImmunoResearch, 1:10.000) was used. For development ECL Western Blotting Substrate (Thermo Scientific, Waltham, MS, United States) was applied followed by luminography. Three independent experiments were performed.

Total cell protein lysates were extracted from transfected 293T cells or infected MDCK cells with CA630 lysis buffer (150 mM NaCl, 1% CA630 detergent, 50 mM Tris base [pH 8.0]). Cellular proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Germany). Each PVDF membrane was blocked with 0.1% Tween 20 and 5% nonfat dry milk in Tris-buffered saline and subsequently incubated with a primary antibody. Primary antibodies were specific for influenza A virus PA (1:3000, GeneTex, USA), influenza A virus PB1 (diluted 1:3000, Thermo Fisher Scientific, USA), influenza A virus PA-X (diluted 1:2000, was kindly provided by Dr. Xiufan Liu, YangZhou University, China). The secondary antibody used was either horseradish peroxidase (HRP)-conjugated anti-mouse antibody or HRP-conjugated anti-rabbit antibody (diluted 1:10,000 Jackson ImmunoResearch USA), as appropriate. HRP presence was detected using a Western Lightning chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany), following the manufacturer's protocol.

Protein was extracted using RIPA buffer (Sigma-Aldrich) for Western Blot. Protein concentration was measured with using BCA assay (DAKO). Primary antibodies for Western blot are rabbit anti-ERK5 (Cell signaling, San Jose, CA, USA), rabbit anti-pERK5 (Cell signaling) and rabbit anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA). Secondary antibody is HRP-conjugated anti-rabbit antibody (Jackson ImmunoResearch Labs). Images shown in the figure were representative from 5 repeats. Densitometry of Western blots was quantified with Image J software (NIH, Bethesda, MA, USA).

Livers were dissected and subsequently frozen in liquid nitrogen. The tissue of each liver was homogenized in a buffer and centrifuged at 14,000×g. Protein (65 μg) from the supernatant of each sample was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in TBST buffer containing 10% non-fat milk for 1 h at room temperature. Immunoblotting was performed by incubating the blocked membrane overnight at 4 °C with a monoclonal mouse leptin receptor antibody (Gene Tex/GTX37636, Irvine, CA, USA), Sirt1 antibody (cell signaling /#9475, Danver, MA, USA), ACE2 antibody (abcam/ab108252, Cambridge, MA, USA), and malondialdehyde (MDA) antibody (abcam/ab27642, Cambridge, MA, USA). The membranes were then incubated with secondary HRP conjugated anti-rabbit antibody (1:5000; Jackson Immuno Research, West Grove, PA USA) or anti-mouse antibody (1:10,000; Jackson Immuno Research, West Grove, PA USA) for 1 h at room temperature. Western blots were visualized using an ECL kit (Perlcin Elmer In./NEL 105001EA, Boston, MA, USA).