DEF cells were lysed with RIPA buffer (beyotime Biotech, Shanghai, China) at 36 h post transfection. Cell lysates were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the isolated proteins were electroblotted on PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% milk for 1.5 h in TBST (0.1% Tween-20 in PBS) and then incubated with mouse anti-His monoclonal antibody (Ruiyingbio, Suzhou, China) or mouse anti-β-actin monoclonal antibody (Transgen Biotech, Beijing, China) for 2 h, in 37℃. HRP-conjugated goat anti-mouse IgG (Transgen Biotech) antibody 1:5000 was used as the secondary antibody, incubate in 37℃ for 1h. The proteins were visualized by chemiluminescence using an ECL kit (Bio-Rad, USA).
Hrp conjugated goat anti mouse igg
Manufactured by Transgene
Sourced in China
HRP-conjugated goat anti-mouse IgG is a laboratory reagent used to detect the presence of mouse immunoglobulin G (IgG) in a sample. It consists of goat-derived antibodies that have been conjugated with the enzyme horseradish peroxidase (HRP). This conjugation allows for the indirect detection of mouse IgG through a colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg, by Transgene (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Mouse anti β actin monoclonal antibody, by Transgene (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab126704, by Abcam (1 mentions)
Ab223868, by Abcam (1 mentions)
Ab155987, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
Easysee western blot kit, by Transgene (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Neomycin, by Merck Group (1 mentions)
Pncmo2, by Takara Bio (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
7 protocols using hrp conjugated goat anti mouse igg
1
Western Blot Analysis of His-tagged Proteins
2
Western Blot Analysis of PRRSV N Protein
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Cellular specimens were harvested and lysed using radio-immunoprecipitation assay buffer comprising 50 mM Tris, pH 7.2, 150 mM NaCl, 1% sodium deoxycholate, and 1% Triton X-100. Following lysate preparation, protein samples were separated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and were subsequently transferred onto nitrocellulose filter membranes (NC, Cytiva, Washington, United States). The purified blots were incubated with mouse anti-PRRSV N protein (N) antiserum (1,500) at 4°C overnight, followed by treatment with HRP-conjugated goat anti-mouse IgG (1,500 in TBST, TransGen, Beijing, China) at 37°C for 50 min. Signals were detected using the ECL chemiluminescent detection system (Tanon, Shanghai, China) per the manufacturer’s instructions, and resultant images were captured using the Western Blotting imaging system (Tanon, Shanghai, China).
3
Western Blot Analysis of DNA Damage Markers
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Cells were directly lysed using 2 × protein lysing buffer (2% SDS, 5% β-mercaptoethanol, 0.5% sucrose and 0.2% bromophenol blue) at 4°C for 20 min, and then the lysates were heated in a metal bath at 98°C for 5 min. Cell lysates were separated on 4–12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked using 5% non-fat milk and immunoblotted using indicated antibodies. The protein signals were detected using a New-SUPER ECL Substrate Kit (Keygen Biotech, Nanjing, China). The primary antibodies used in this study targeted phospho-ATR (S428) (1:1000, abcam, ab178407), phospho-ATM (S1981) (1:1000, Cell Signaling Technology, #5883), phospho-p53 (S15) (1:1000, abcam, ab223868), phospho-histone H2AX (S139) (1:1000, Cell Signaling Technology, #9508), GCLM (1:1000, abcam, ab126704), HO-1 (1:1000, abcam, ab68477), phospho-p38 (T180/Y182) (1:500, Santa Cruz Biotechnology, sc-17852-R), phospho-Hsp27 (S82) (1:1000, abcam, ab155987), phospho-ATF2 (T71) (1:1000, abcam, ab32019) and Actin (1:3000, Bioss, #bs-0061R). The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000, TransGen Biotech, Beijing, China, #HS201-01) and HRP-conjugated goat anti-rabbit IgG (1:10,000, TransGen Biotech, Beijing, China, #HS101-01).
4
Analyzing MAPK and NF-κB Signaling in GM-DCs
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For analyzing the activation of MAPK and NF-κB signaling pathways, GM-DCs were treated with 102L.L and 103L.L for 30 min or 120 min and proteins were extracted by nucleoprotein and cytoplasmic protein extraction kit (Biorebo Biotech, China). 40 ng/mL LPS (Sigma-Aldrich, USA) was used as the positive control.
For detection of EGFP and OVA expression, the recombinant L.L with pNZ8149-penp-OVA (L.L-OVA) was cultured at 30°C to OD595nm of 0.2-0.6. Then 10 ng/ml nisin was added and cultured for 0 h, 9 h, 12 h and 24 h at 30°C. The recombinant L.L with pMG36e is auto-inducible. The recombinant L.L was harvested by centrifugation (12000 rpm, 10 min), and the proteins were obtained by liquid nitrogen grinding. The expression of related proteins was detected by Western blot, including EGFP (TransGen Biotech, China) and OVA (Elabscience, China). After incubation with HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-rabbit IgG (TransGen Biotech, China), the target proteins were detected using EasySee Western blot kit (TransGen Biotech, China).
For detection of EGFP and OVA expression, the recombinant L.L with pNZ8149-penp-OVA (L.L-OVA) was cultured at 30°C to OD595nm of 0.2-0.6. Then 10 ng/ml nisin was added and cultured for 0 h, 9 h, 12 h and 24 h at 30°C. The recombinant L.L with pMG36e is auto-inducible. The recombinant L.L was harvested by centrifugation (12000 rpm, 10 min), and the proteins were obtained by liquid nitrogen grinding. The expression of related proteins was detected by Western blot, including EGFP (TransGen Biotech, China) and OVA (Elabscience, China). After incubation with HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-rabbit IgG (TransGen Biotech, China), the target proteins were detected using EasySee Western blot kit (TransGen Biotech, China).
5
Recombinant LOX-1 Protein Expression
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B. choshinensis SP3 (Takara, Japan) was used for protein expression. The B. choshinensis-E. coli shuttle vector pNCMO2 (Takara) was used for expression in B. choshinensis. A site-directed mutagenesis kit was purchased from Stratagene (Shanghai, China). Recombinant human LOX-1 protein was purchased from Sino Biological Inc., China. Anti-His tag antibody and HRP-conjugated goat anti-mouse IgG were purchased from TransGen Biotech (Beijing, China). Bovine serum albumin (BSA) and neomycin were purchased from Sigma-Aldrich, UK. TM medium with modifications (3% glucose, 2.2% polypeptone, 0.8% meat beef extract, 0.2% yeast extract, 0.001% FeSO4·7H2O, 0.001% MnSO4·4H2O, and 0.0001% ZnSO4·7H2O) was used to culture the B. choshinensis strains.
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B. choshinensis SP3 (Takara, Japan) was used for protein expression. The B. choshinensis-E. coli shuttle vector pNCMO2 (Takara) was used for expression in B. choshinensis. A site-directed mutagenesis kit was purchased from Stratagene (Shanghai, China). Recombinant human LOX-1 protein was purchased from Sino Biological Inc., China. Anti-His tag antibody and HRP-conjugated goat anti-mouse IgG were purchased from TransGen Biotech (Beijing, China). Bovine serum albumin (BSA) and neomycin were purchased from Sigma-Aldrich, UK. TM medium with modifications (3% glucose, 2.2% polypeptone, 0.8% meat beef extract, 0.2% yeast extract, 0.001% FeSO4·7H2O, 0.001% MnSO4·4H2O, and 0.0001% ZnSO4·7H2O) was used to culture the B. choshinensis strains.
6
Screening Hybridomas for PDCoV N Protein-Specific mAbs
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To establish an ELISA for screening hybridomas secreting mAbs specific to the N protein of PDCoV, a standard method was employed. Briefly, the recombinant PDCoV N protein (5 µg/ml) was coated onto 96-well ELISA plates. The coated plates were washed three times with 0.01 M PBS (pH 7.2) and then blocked with 5% skimmed milk. After three times washing, the plates were incubated with the sera of mice who had been immunized with the recombinant PDCoV N protein. Subsequently, the plates were incubated with HRP-conjugated goat anti-mouse IgG (1:2000, TransGen Biotech). The unbound secondary antibody was washed off with PBS. Signal reaction was activated utilizing 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, and then stopped with 2 M H2SO4, after which the absorbance was read at OD450. Tested samples that gave an absorbance value greater than 0.080 were defined as positive.
7
Indirect ELISA for IgG Detection
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An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect IgG levels in the sera of immunized mice. At 4°C overnight, 96-well plates were coated with 5 μg/100 μL of purified
recombinant protein diluted in PBS per well (To determine IgG levels of the inactivated vaccine group, each well was coated with 4 × 109 CFU/100 μL of inactivated G.
parasuis CY1201 strain diluted in PBS). The wells were washed thrice with PBST (PBS containing 0.05% Tween-20) and then blocked with 5% (w/v) skim milk in PBST for 2 hr at 37°C.
The plates were washed for thrice and incubated with 100 μL of sera diluted in 1:400 for 1 hr at 37°C. After three washes, HRP-conjugated goat anti-mouse IgG (TransGen Biotech), diluted
1:3,000 in PBS, was used as the secondary antibody and incubated for 1 hr at 37°C. The plates were washed thrice with PBST, and 100 μL of TMB was added to each well, followed by incubation
at room temperature for 30 min in the dark. The reaction was stopped by adding 50 μL of 2 M H2SO4 in each well, and OD450 was measured using a microplate
reader (Bio-Rad, Hercules, CA, USA). Simultaneously, the serum tested on day 14 after the second immunization was diluted 100–204,800 times with PBS as the primary antibody, and the antibody
titer was determined.
recombinant protein diluted in PBS per well (To determine IgG levels of the inactivated vaccine group, each well was coated with 4 × 109 CFU/100 μL of inactivated G.
parasuis CY1201 strain diluted in PBS). The wells were washed thrice with PBST (PBS containing 0.05% Tween-20) and then blocked with 5% (w/v) skim milk in PBST for 2 hr at 37°C.
The plates were washed for thrice and incubated with 100 μL of sera diluted in 1:400 for 1 hr at 37°C. After three washes, HRP-conjugated goat anti-mouse IgG (TransGen Biotech), diluted
1:3,000 in PBS, was used as the secondary antibody and incubated for 1 hr at 37°C. The plates were washed thrice with PBST, and 100 μL of TMB was added to each well, followed by incubation
at room temperature for 30 min in the dark. The reaction was stopped by adding 50 μL of 2 M H2SO4 in each well, and OD450 was measured using a microplate
reader (Bio-Rad, Hercules, CA, USA). Simultaneously, the serum tested on day 14 after the second immunization was diluted 100–204,800 times with PBS as the primary antibody, and the antibody
titer was determined.
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