Protein samples were separated by 10% SDS-PAGE, followed by transferring to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Cat ISEQ00010). After blocking with 5% milk, the membranes were incubated with the primary antibodies mouse anti-Flag antibody (1:3000) (Proteintech, Cat 66008-3-Ig), mouse anti-Myc antibody (1:3000) (Proteintech, Cat 60003-2-Ig), mouse anti-Rab18 antibody (1:500) (Santa Cruz Biotechnology, Cat sc-393168), mouse anti-GST antibody (1:2000) (Abcam, Cat ab19256), or rabbit anti-actin antibody (1:5000) (Abcam, Cat ab8227) at 4°C for 12 h. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech, Cat SA00001-1) secondary antibody (1:5000) for 2 h. Finally, the signal was detected using an enhanced chemiluminescence (ECL) western blot analysis system. β-actin served as an internal control protein.
Horseradish peroxidase hrp conjugated goat anti mouse igg
Manufactured by Proteintech
Sourced in United States, China
Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG is a secondary antibody used in various immunoassay techniques. It is produced by conjugating HRP, an enzyme, to goat-derived antibodies specific for mouse immunoglobulin G (IgG). This conjugate allows for the detection and visualization of target proteins or antigens in samples containing mouse IgG.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Rabbit anti actin antibody, by Abcam (1 mentions)
Mouse anti flag antibody, by Proteintech (1 mentions)
Mouse anti myc antibody, by Proteintech (1 mentions)
Mouse anti gst antibody, by Abcam (1 mentions)
Diaminobenzidine, by Boster Bio (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Trans blot transfer cell, by Bio-Rad (1 mentions)
Stop solution for tmb substrate, by Beyotime (1 mentions)
Mouse anti puromycin monoclonal antibody, by Merck Group (1 mentions)
Mouse anti β actin monoclonal antibody, by Proteintech (1 mentions)
Tmb substrate, by Avantor (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Transblotting apparatus, by Bio-Rad (1 mentions)
His tag 4c2 monoclonal antibody, by Bioworld Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Dms114, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
12 protocols using horseradish peroxidase hrp conjugated goat anti mouse igg
1
Western Blot Analysis of Protein Samples
2
Immunoblotting of recombinant CsHK protein
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Purified rCsHK (2 µg) and total worm extract (30 µg) were subjected to 12% SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Whatman, UK) at 100 V for 1 h in a Trans-Blot transfer cell (Bio-Rad, USA). The PVDF membranes were blocked with 5% (w/v) skimmed milk in phosphate buffer saline (PBS, pH 7.4) overnight at 4°C and then probed with a mouse anti-His tag monoclonal antibody (1∶2,000 dilution, Novagen, USA), mouse anti-rCsHK serum (1∶2,000 dilution) or serum from a pre-immune mouse (1∶2,000 dilution) for 2 h at room temperature. After washing with PBS 3 times, the membranes were incubated in horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1∶2,000 dilution, Protein tech., USA) for 1 h at room temperature. Both the primary and secondary antibodies were diluted with 0.1% BSA in PBS (pH 7.4). After washing 5 times, the membranes were finally developed in color with diaminobenzidine (DAB, Boster, China) reagents according to the manufacturer’s instructions.
3
ELISA for Detection of p30 Protein Antibodies
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Purified recombinant p30 protein constructs were coated on flat-bottom polystyrene plates (1 μg/ml; 100 μl/well) in carbonated coating buffer (pH 9.6) and incubated overnight at 4°C. The plate was washed five times with PBST (0.05% Tween in PBS, v/v), and the plate was blocked with 5% skimmed milk in PBS, for 1 h at 37 °C. After washing the plates as above, 50 μl undiluted hybridoma supernatants was added. Positive serum from mice immunized with p54 recombinant protein and negative serum from unimmunized mice, diluted 1:10,000, were also included in duplicate as a control. The plate was incubated for 30 min at 37°C, and a washing step was repeated. Thereafter, horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Proteintech Group, Inc., Rosemont, IL, USA) diluted 1:10,000 was added and incubated for 30 min at 37°C. Following washing five times, reaction was developed by adding a chromogenic substrate solution (TMB) (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 10 min and stopped with Stop Solution for TMB Substrate (Beyotime Biotechnology Co., Ltd., Shanghai, China). The plates were read at 630 nm.
4
Antibody Validation for Ribosomal Proteins
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The following antibodies were used in this study: mouse anti-hemagglutinin (HA) monoclonal antibody (Proteintech); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech); mouse anti-puromycin monoclonal antibody (Millipore); goat anti-mouse IgG polyclonal antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific); mouse anti-β-actin monoclonal antibody (Proteintech); rabbit anti-RPS6 polyclonal antibody (ABclonal); rabbit anti-RPS18 polyclonal antibody (ABclonal); rabbit anti-RPS2 polyclonal antibody (ABclonal); rabbit anti-RPS3 polyclonal antibody (ABclonal); rabbit anti-RPS8 polyclonal antibody (ABclonal); rabbit anti-RPS9 polyclonal antibody (ABclonal); rabbit anti-RPL30 polyclonal antibody (ABclonal).
5
PEDV S1 Protein Immunoreactivity Assay
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Indirect ELISA was used to identify the immune reactivity of the truncated proteins and the screen of positive hybridoma cells. The ELISA plates were plated with purified PEDV S1 protein or synthesized peptides (400 ng/well) in carbonate bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3 [pH 9.6]) and coated at 4 °C overnight. The plates were blocked for 1 h at 37 °C using 5% non-fat dry milk in phosphate buffer with 0.05% Tween-20 (PBST). After being washed thrice, the plates were incubated with 100 μL diluted anti-sera or antibodies at 37 °C for 1 h. The plates were incubated with horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG (Proteintech Group, China) with 1:20,000 dilution in PBST at 37 °C for 1 h after being washed thrice in PBST. Then, plates were washed with PBST and incubated with 50 μL/well of TMB liquid (Amresco, Solon, Ohio, USA) for 15 min at room temperature with protection from light. The results were read with OD450 values after being stopped by 2 M H2SO4 (50 μL/well).
6
Indirect ELISA for HLA-F Reactivity
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Indirect ELISA was used to determine the immune reactivity of HLA-F and to screen positive hybridoma cells as described previously 28 (link). Briefly, ELISA plates were plated with purified HLA-F protein in PBS (pH 7.4), coated at 4 °C overnight and. After which, the plates were incubated with 100 μL diluted antibodies at 37 °C for 1 h, and followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech Group, China) at a 1:5000 dilution in PBST at 37 °C for 1 h. After washing, 50 μL/well of TMB substrate (Amresco, Solon, Ohio, USA) was added into the wells, and the plates were incubated for 15 min at room temperature in the dark. OD450 values were obtained after stopping the reaction with 1 M HCl (100 μL/well).
7
T Cell Activation Immunophenotyping Protocol
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Anti-CD16/32 (2.4G2), anti-CD3(145-2c11), anti-CD28 (37.51), FITC-anti-MHC II (M5/114.15.2), FITC-anti-CD69 (H1.2F3), PE-labeled anti-CD4 (GK1.5), APC-labeled anti-CD8 (53-6.7), biotin-labeled anti-TCRβ (H57-597), and PE-Cy5-labeled anti-TCRβ (H57-597) were obtained from e-Bioscience; anti-GAPDH, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated donkey anti-human IgG were obtained from proteintech; additional biotin-conjugated lens culinaris agglutinin (LCA) were purchased from Vector; anti-TCRαβ (ab25336), anti-pZAP70 (ab194800), anti-ZAP70 (ab32410), Natural streptavidin protein (FITC) (ab136201), and streptavidin (HRP) (ab7403) were purchased from Abcam.
8
Western Blot Protein Analysis
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Pretreated bacteria or purified protein were solubilized and loaded onto SDS polyacrylamide gels. After electrophoresis, samples were stained with Coomassie brilliant blue, or transferred (2 h at 100 V) to a polyvinylidene difluoride membrane (Roche Diagnostics, German) using a transblotting apparatus (Bio-Rad, USA). The membrane was blocked in 5 % skimmed milk-TBST at 4℃ overnight. The membrane was incubated with His-tag (4C2) monoclonal antibody (1:8,000; Bioworld Technology, China) at RT for 1 h followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:20,000; Proteintech, China) at RT for 1 h, and visualized with enhanced chemiluminescence (CWBio, China).
9
Protein Expression Analysis in Cell Lines
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Proteins from whole cell lysates of L929, CHO, MCF7, HEK293, H526, A549, HeLa, U-87MG, and DMS114 (all purchased from ATCC; Fig. S1 ) were obtained using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) with a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Proteins at 10 µg/lane were loaded onto a 12% acrylamide gel, resolved using SDS-PAGE; and were then transferred to a polyvinylidene difluoride membrane (Merck KGaA). After blocking with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 2 h at room temperature, primary antibodies against MCT1, MCT4, CD147 (1:1,000; cat. no. ab119020; Abcam), or matrix metalloproteinase (MMP)2 (1:2,000; cat. no. 66366-1-Ig; ProteinTech Group, Inc.) were incubated overnight at 4°C and then washed with PBS-Tween20 (0.05% Tween-20) and incubated with the secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000; cat. no. A-31430; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Enhanced chemiluminescence (ECL) reagent (Vazyme Biotech Co., Ltd.) was used to detect the proteins, and the immunoblots were developed using the Image Scanner ChemiDoc MP (Bio-Rad Laboratories, Inc.) and analyzed by ImageJ v1.5.1 (National Institutes of Health).
10
m6A RNA Methylation Analysis
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The poly(A)+ RNAs were first denatured by heating at 65°C for 5 min and transferred onto a nitrocellulose membrane (Amersham; GE Healthcare, USA) with a Bio-Dot apparatus (Bio-Rad Laboratories, USA). The membranes were then UV crosslinked, blocked, incubated with m6A antibody (1:1,000; Abcam, USA) overnight at 4°C, and subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:3,000; Proteintech Group, USA). Finally, the membranes were visualized using the chemiluminescence system (Bio-Rad Laboratories, USA). The membrane stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) was used to ensure consistency among the different groups.
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