Anti mouse igg hrp antibody

For sample preparation for SDS-PAGE, soluble proteins were mixed with ×3 SDS-PAGE sample buffer and denatured by heating at 99°C for 5 min. Denaturation of membrane proteins and cell extracts was conducted at 37°C for 30 min. After separation by SDS-PAGE using e-PAGEL 5–20% gel (ATTO), proteins were transferred to an Immobilon-P membrane (Millipore) using EzFastBlot (ATTO). After blocking with 5% skim milk solubilized in Tris-buffered saline containing 0.01% Tween20 (TBST) for 1 h, the membrane was treated for 1 h by primary antibodies at the appropriate concentration. The membrane was washed 3 times with TBST, and treated with the appropriate secondary antibody, anti-rabbit IgG-HRP or anti-mouse IgG-HRP antibody (GE Healthcare). After TBST washing for three times, the antibody was visualized using Immunostar LD (Wako). Chemiluminescent signal was detected by ImageQuant LAS 4000 imager (GE Healthcare).

Protein concentration of the whole‐cell lysate was determined with the DC protein assay kit (Bio‐Rad Cat. 500‐0111). Whole‐cell lysates were mixed with equal volumes of 2× SDS protein loading buffer (100 mM Tris–HCl, pH 6.8; 4% SDS; 0.2% bromophenol blue; 20% glycerol; 10 mM DTT) and boiled at 95°C for 5 min. 2 μg of protein was loaded, followed by SDS–PAGE on a 12% polyacrylamide gel with cathode buffer (0.1 M Tris, 0.1 M tricine, 0.1% SDS) on top of the wells and anode buffer (0.2 M Tris/Cl, pH 9.9) in the bottom. After electrophoresis, samples in the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane as described (Minnen et al, 2011). Teichoic acid was detected with anti‐PC specific monoclonal antibody TEPC‐15 (M1421, Sigma) by 1:1,000 dilution as first antibody, and then with anti‐mouse IgG HRP antibody (GE Healthcare UK Limited) with 1:5,000 dilution as second antibody. The blots were developed with ECL prime Western blotting detection reagent (GE Healthcare UK Limited), and the images were obtained with a Bio‐Rad imaging system.

Humoral responses were analyzed in pooled mice plasma. Binding antibodies to HIV-1 Gag were detected by Western immunoblot using cell extracts from HEK293 cells transfected with 1 μg of Gag and Gag-Pol expression vectors, separated on 12% SDS-PAGE, and the membranes were probed with pooled plasma (at a 1:100 dilution) and the bands were visualized by anti-mouse IgG-HRP antibody (1:10,000 dilution, GE Healthcare, Piscataway, NJ). Serial dilutions of plasma samples were analyzed by standard HIV-1 clade B p24^gag ELISA (Advanced Bioscience Lab, Rockville, MD), measuring optical absorbance at 450 nm.

Immunoblot analysis was performed as previously described^{10 (link)}. Recombinant proteins or cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with Blocking One solution (Nacalai Tesque), the membranes were incubated with monoclonal mouse anti-Flag M2 antibody (Sigma-Aldrich, F1804) and AK97³³ , followed by incubation with respective horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-mouse IgG antibody-HRP (GE Healthcare) and anti-mouse IgM antibody-HRP (ENZO), respectively. The hexahistidine (His₆)-tagged and biotin-labeled recombinant proteins were detected using anti-His₆-Peroxidase (Roche) and HRP-conjugated Streptavidin (Sigma), respectively. The protein bands were developed with Immobilon Western Chemiluminescent HRP substrate solution (Millipore) and were imaged with an Amersham™ Imager 600 (GE Healthcare). After removing the antibodies or avidin by Western Blot Stripping Buffer (Takara Bio Inc.), the membrane was reproved using different antibodies. For deglycosylation, lysates were incubated with 50 units/mL of PNGase F (New England BioLabs) at 37 °C for 48 h before being subjected to SDS-PAGE. Densitometry analysis was performed using Amersham™ Imager 600 Analysis Software (GE Healthcare).

Western blots were performed according to methods in previous studies^{29 (link),30 (link)}. At 72 h post transfection, the EPO and sFγRIII recombinant proteins were purified with anti-Flag M2 Affinity Gel (Sigma-Aldrich). The gel was washed twice with phosphate-buffered saline, and proteins were eluted with the 3× FLAG tag peptide (Sigma-Aldrich). The purified proteins were subjected to 3–10% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). After blocking with Blocking One solution (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with monoclonal mouse anti-Flag M2 (Sigma-Aldrich, F1804), anti-human LMAN1 (ERGIC-53) (Proteintech, Tokyo, Japan, 13364), anti-human SDNSF/MCFD2 (R&D System, Minneapolis, MN, MAB2357), and anti-β-actin (Sigma-Aldrich, A2228) antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibody [anti-mouse IgG antibody-HRP, GE Healthcare (Piscataway, NJ) and anti-rabbit Ig antibody-HRP, Cell Signaling Technology (Danvers, MA)] in 20 mM Tris-HCl (pH 7.6) containing 150 mM NaCl and 0.05% Tween-20. The protein bands were visualized with Immobilon Western Chemiluminescent HRP substrate solution (Millipore).