Horseradish peroxidase hrp conjugated anti rabbit igg

Full CDS for TpmII variants 3, 4, 7 and 8 were amplified by PCR from the recombinant plasmids (see Section 2.4) using specific primers with appended restriction sites (Supplementary Table S3). Amplicons were ligated into the pGEX-KG bacterial expression vector between BamHI and XbaI restriction sites (TpmII.3, 4 and 8) or between XbaI and XhoI sites (TpmII.7). Constructs were sequenced to verify inserted sequences. GST-TpmII fusion proteins (5′ GST) were expressed in Escherichia coli, isolated by affinity chromatography on glutathione–agarose (GE Healthcare, UK), reapplied to glutathione–agarose in PBS containing 1% Triton X-100 (Sigma–Aldrich, UK), washed with PBS and then cleaved at 22 °C for 16 h with 50 units (U) of thrombin per mg of fusion protein to release free TpmII.3, 7 or 8 and 4 h with 30 U of thrombin per mg of fusion protein to release TpmII.4. In each case the absence of contaminating GST was confirmed by ELISA using rabbit-anti-GST antisera (Sigma) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma).

Chromatography fractions and purified proteins were electrophoresed on SDS-PAGE and visualized by Coomassie Brilliant Blue R-250 (Sigma Aldrich, USA). For western blotting, purified proteins were subjected to 15% (w/v) SDS-PAGE and electro transferred onto nitrocellulose membrane (Bio-Rad Laboratories, CA, USA). Membranes were blocked with 5% bovine serum albumin (Sigma Aldrich, USA) in PBST (1X PBS in 0.05% Tween-20) for 2 hours at room temperature and incubated overnight with diluted anti-His and anti-GST antibodies (Sigma Aldrich, USA) in blocking buffer (1:1000). The blots were washed with PBST and incubated with horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Sigma Aldrich, USA) diluted in PBST (1:10,000). After subsequent washing, the blots were developed with 1 mg/ml 3,3’-diaminobenzidine (DAB) (Sigma Aldrich, USA) and 1 μl/ml hydrogen peroxide in phosphate buffer saline.

Purified TcaB was biotin labeled using a EZ-Link sulfo-N-hydroxysuccinimide (NHS) liquid chromatography (LC) biotinylation kit (Thermo Fisher Scientific) by following the manufacturer’s protocol. Next, 5 µg of purified GmCAD fragments dissolved in loading buffer were heat-denatured and separated in 4–20% gradient SDS-PAGE gel (BioRad) and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The transblotted membrane was blocked overnight in PBST (PBS containing 0.05% Tween-20) and incubated with biotinylated TcaB (5 µg mL⁻¹) in blocking buffer for 1 h at 28°C. After three washes (10 min each) with PBST, the membrane was probed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma-Aldrich) in blocking buffer (1: 10,000 dilution) for 1 h at 28°C. After three washes, the membrane was developed using a ECL chemiluminescence western blotting kit (BioRad).

Antibody verification was performed using Western blot. The laboratory-preserved Sjp90α prokaryotic expression protein was separated by SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, MA, USA) at 300 mA for 60 min. The SEA egg antigen was used as a positive control. The membranes were blocked with 5% skim milk in TBST (25 mmol/L Tris base, 150 mmol/L NaCl, and 0.1% Tween 20) for 2 h and incubated overnight at 4 °C with the Sjp90α-1-peptide-based antibody (1:1000). The membranes were washed with TBST and probed with horseradish peroxidase (HRP)-conjugated antirabbit IgG (Sigma-Aldrich) (1:5000) for 1 h at RT. The blots were developed with an enhanced chemiluminescence (ECL) system (Merck Millipore), and images were photographed using a ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, CA, USA).

The protein expression of cyclin-dependent kinases (CDKs) and cyclins was analyzed using western blot assays. Primary rabbit polyclonal antibodies against CDKs and cyclins were purchased from Sigma-Aldrich. The secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma-Aldrich). After homogenization using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Wuhan, China), the proteins from tissues or cells were extracted and quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology). For each sample, 10 µg total proteins were electrophoresed on 15% sodium dodecyl sulfate-polyacrylamide gel and transferred to a poly-vinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was then blocked using 4% skimmed milk for 1 h at room temperature and incubated with primary antibodies against CDK1 (1:1000), CDK2 (1:1000), CDK4 (1:800), cyclin (1:500), cyclin B (1:1000) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000) at 4°C overnight. After washing with Tris-buffered saline and 84 Tween 20 (TBST) buffer (pH 7.6, 20 mM Tris-HCl, 137 mM NaCl, 0.01% Tween-20), the membranes were incubated with secondary antibody and visualized using enhanced chemiluminescence reagents (ECL; EMD Millipore).

Preparation of the parasite lysate and Western blot analysis was performed following the method described earlier [58 (link)]. K133WT and K133AS-R cell lysates (100 µG) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membrane strips were blocked and incubated sequentially with anti-AQP1 (1:1000), anti-HSP70 (1:500), or anti-tubulin (1:1000) (endogenous control) primary antibodies. Following this, the membrane was probed with Horse radish Peroxidase (HRP)-conjugated anti-rabbit IgG (1:80,000) produced in mice (Sigma Aldrich, St. Louis, MO, USA). Blot was developed using Western blot detection enhanced chemiluminescence (ECL) detection reagent (Merck, Burlington, MA, USA). The image was scanned with ChemiDoc (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab™ 5.1 software (Bio-Rad, Hercules, CA, USA) [58 (link)].