Co-IP was performed to confirm the interaction between two proteins. The cellular protein was extracted from H9c2 cells using RIPA lysis buffer supplemented with 1% PMSF (Beyotime Biotech). After protein concentration determination, Co-IP was performed using a Co-IP kit (Thermo Scientific) according to the manufacturer’s instruction. Briefly, the antibody against GSK3β (ProteinTech) or flag tag (ProteinTech) was immobilized to AminoLink resin by incubating in sodium cyanoborohydride solution (75 mM), and the resin was pre-cross-linked to spin column. The protein samples were incubated with the antibody-resin complex in the tube for immunoprecipitation. Subsequently, the immunoprecipitate was eluted with elution buffer, and used for western blot to detect the content of GSK3β, WSB1, ubiquitin and GAPDH. The information of antibodies was shown as follows: rabbit anti-GSK3β (1:5000; ProteinTech), rabbit anti-WSB1 (1:1000; Thermo Scientific), rabbit anti-ubiquitin (1:5000; ProteinTech), mouse anti-flag tag (1:5000; ProteinTech), rabbit anti-HA tag (1:5000; ProteinTech), goat anti-rabbit IgG-HRP (1:3000; Solarbio), goat anti-mouse IgG-HRP (1:3000; Solarbio).
Goat anti mouse igg hrp
Manufactured by Solarbio
Sourced in China
Goat anti-mouse IgG-HRP is a secondary antibody conjugated to horseradish peroxidase (HRP). It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays and Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Solarbio (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Co ip kit, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Rabbit anti ubiquitin, by Proteintech (1 mentions)
Mouse anti flag tag, by Proteintech (1 mentions)
Rabbit anti ha tag, by Proteintech (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Goat anti rabbit igg hrp antibody, by Solarbio (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Anti histone h3, by Beyotime (1 mentions)
Mouse anti ampk alpha 1 ampk alpha 2 antibody, by Abcam (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Rhod 2 am, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Solarbio (1 mentions)
12 protocols using goat anti mouse igg hrp
1
Co-immunoprecipitation of GSK3β Interactome
2
Alanine Scanning of Peptide Epitopes
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Alanine mutation was used to determine the crucial sites of amino acids of peptides S19.2 and S49.4. The amino acid sequence of the peptides was substituted with alanine (A) one by one. At the same time, the alanine (A) in the sequence was substituted with glycine (G). The coupled peptides were coated on the 96-well ELISA plates, peptide S19.2 and peptide S49.4 were used as a positive control in their mutant groups. The monoclonal antibodies 4B, 7D, and 8F, 3D were used as primary antibodies, and the Goat Anti-Mouse IgG/HRP (Solarbio, Cat No. SE131, China) was used as the secondary antibody.
3
Western Blotting Analysis of Insect Proteins
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For Western blotting, equal amounts of protein extracts from whole insects were separated by SDS-PAGE and transferred onto PVDF. The membranes were blocked, incubated with the corresponding primary antibody. The primary antibodies for western blots were diluted with 5% skim milk in 0.1% Tween/TBS for anti-TcAK1 (1:1,000), anti-TcAK2 (1:2,000), anti-TcFOXO (1:1,000), anti-TcAMPKα (1:1,000), anti-α-tubulin (1:5,000) (Proteintech Group, Inc., Chicago, IL, USA), anti-Histone H3 (1:5,000) (Beyotime, Shanghai, China), Mouse Anti-AMPK alpha 1 + AMPK alpha 2 antibody (1:1,000), Recombinant Anti-GST antibody (1:5,000) (Abcam, Cambridge, MA, USA). The specificity of the TcAK1, TcAK2, TcFOXO and TcAMPKα antibodies was verified in our previous studies [29 (link), 58 (link), 59 (link)] The membranes were washed and incubated with the corresponding secondary antibody such as Goat anti-mouse IgG/HRP (1:10,000) (Solarbio, Beijing, China) and Goat anti-rabbit IgG/HRP antibody (1:10,000) (Solarbio, Beijing, China). The blot signals were detected using Tanon High-sign ECL Western Blotting kit (Tanon, Shanghai, China) with a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) as described previously [29 (link)].
4
Evaluation of A2E-induced Retinal Damage
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Dulbecco's modified Eagle's medium (DMEM) and trypsin-EDTA solution were obtained from HyClone (Logan, Utah, USA). Fetal bovine serum (FBS) was obtained from MRC (New Zealand). Hank's balanced salt solution (HBSS), Rhod-2/AM, Fluo-3/AM, and LysoTracker Red DND-99 were obtained from Thermo Fisher Scientific (Waltham, MA). CaTM-2/AM was obtained from Goryo (Sapporo, Japan). NFD and F-127 were obtained from Sigma-Aldrich (St. Louis, MO). Mouse anti-human keratin antibody, goat anti-mouse IgG-HRP, mitochondrial membrane potential assay kit with JC-1, phosphate-buffered saline (PBS), and penicillin-streptomycin were obtained from Solarbio (Beijing, China). Dimethyl sulfoxide (DMSO) was obtained from MP Bio (California, USA). A2E was donated by Sun Xiaodong (The First Affiliated Hospital of Shanghai Jiaotong University).
5
Sensitive Immunoassay Development Protocol
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1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enrofloxacin (ENR), ofloxacin (OFL), lomefloxacin (LOM), N,N-dimethylformamide (DMF), bovine serum albumin (BSA), HAuCl4·4H2O, citrate sodium, and ovalbumin (OVA) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). goat anti-rabbit IgG-HRP, goat anti-rabbit IgG, goat anti-mouse IgG-HRP, goat anti-mouse IgG, 3,3′,5,5′-tetramethylbenzidine solution (TMB), protein-A affinity column, and Tween- 20 were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Rabbit anti-ENR polyclonal antibody, mouse anti-OFL polyclonal antibody, mouse anti-LOM polyclonal antibody, and mouse anti-amide group polyclonal antibody were prepared by our laboratory in the previous studies (The immunogens of these antibodies were respectively ENR-cOVA, OFL-cOVA, LOM-cOVA, and cOVA, the titers of antisera were all over than 200,000). Monoclonal antibody specific to ENR was purchased from Wuhan Huamei Tech. Co., Ltd. (Wuhan, China). All other reagents were analytical or higher grade.
Detailed formulas of all solutions are given in Supporting Information.
Detailed formulas of all solutions are given in Supporting Information.
6
Protein Extraction and Western Blot Protocol
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Total protein was extracted using RIPA lysate and PMSF. The protein concentration was determined by BCA Protein Assay Kit (Solarbio, China). Polyacrylamide gel is consisted of 5% stacking gel and 12% and 14% separating gel. SDS-PAGE was performed to separate the target protein. Then the proteins were transferred to PVDF membrane, which was subsequently blocked with blocking solution (Solarbio, China). Incubation of primary antibody was conducted at 4℃ overnight and secondary antibody incubation was performed at 37℃ for 1 h. After incubation of antibodies, the protein bands were detected by ECL-based chemiluminescence reaction. The optical density of the target band was analyzed by Gel-Pro-Analyzer software (USA). The antibodies were as follows: HPGD antibody (1:500 dilution, A6926, Abclonal, China); Nr4a1 antibody (1:2000 dilution, 12235-1-AP, proteintech, China); GAPDH (1:10000 dilution, 60004-1-Ig, proteintech, China); Goat anti-rabbit IgG-HRP (1:3000 dilution, SE134, Solarbio, China); and Goat anti-mouse IgG-HRP (1:3000 dilution, SE131, Solarbio, China).
7
Protein Extraction and Western Blot Analysis
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Protein was isolated from the cells using the radioimmunoprecipitation
assay (RIPA) buffer. The concentration of protein was determined using
the Pierce BCA Protein Assay Kit (Cat# 23225, Thermo Fisher Scientific).
Protein (20–40 μg) was loaded in the wells of Nu-Page
precast 4–10% sodium dodecyl sulfate (SDS)-poly-acrylamide
gels (Invitrogen) and then transferred onto poly(vinylidene fluoride)
membranes by a dry electroblotting method using an I-Blot. The membranes
were then incubated with specific primary antibodies including c-Jun
antibody (#9165, Cell Signaling Technology), c-Fos antibody (#2250,
Cell Signaling Technology), NF-κB p65 (#3034, Cell Signaling
Technology), secondary antibodies including mouse anti-rabbit IgG-HRP
(Santa Cruz, sc-2357, Santa Cruz Biotechnology), and Goat Anti-Mouse
IgG-HRP (SE131, Solarbio) step by step and incubated with visualized
SuperSignal West Atto Ultimate Sensitivity Substrate (Cat# A38555,
Thermo Fisher Scientific) for 5 min before visualizing on the X-ray
film (no. C500046, Sango Biotech) in the darkroom.
assay (RIPA) buffer. The concentration of protein was determined using
the Pierce BCA Protein Assay Kit (Cat# 23225, Thermo Fisher Scientific).
Protein (20–40 μg) was loaded in the wells of Nu-Page
precast 4–10% sodium dodecyl sulfate (SDS)-poly-acrylamide
gels (Invitrogen) and then transferred onto poly(vinylidene fluoride)
membranes by a dry electroblotting method using an I-Blot. The membranes
were then incubated with specific primary antibodies including c-Jun
antibody (#9165, Cell Signaling Technology), c-Fos antibody (#2250,
Cell Signaling Technology), NF-κB p65 (#3034, Cell Signaling
Technology), secondary antibodies including mouse anti-rabbit IgG-HRP
(Santa Cruz, sc-2357, Santa Cruz Biotechnology), and Goat Anti-Mouse
IgG-HRP (SE131, Solarbio) step by step and incubated with visualized
SuperSignal West Atto Ultimate Sensitivity Substrate (Cat# A38555,
Thermo Fisher Scientific) for 5 min before visualizing on the X-ray
film (no. C500046, Sango Biotech) in the darkroom.
8
Western Blot Analysis of Protein Expression
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Cells and tissues were lysed in RIPA buffer (Solarbio) containing protease and phosphatase inhibitors. BCA Protein Assay kit (Beyotime) was used to measure the concentration of protein. Protein samples were separated by SDS-PAGE at 80 V for 2.5 h and then transfected into PVDF membranes (Millipore, MA, USA) for 2 h. After blocking in 5% fat-free milk for 1.5 h, the membranes were incubated with primary antibodies at 4℃ overnight (see Table. S1 for primary antibodies information). Next day, the membranes were incubated with secondary antibodies for 2 h: goat anti-rabbit IgG-HRP (Solarbio; SE134; 1:3000); goat anti-mouse IgG-HRP (Solarbio; SE131; 1:3000). Finally, the membranes were washed in TBST and detected by ECL chemiluminescent reagent (Solarbio).
9
Quantifying Antigen-Specific IgG via ELISA
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Serum samples were examined for antigen-specific immunoglobulin G (IgG) by ELISA. ELISA was performed according to a previous study [18 (link)]. Briefly, 96-well plates were coated with SLS and fimbriae proteins (F4, F5, F6, F18 and F41), kept overnight at 4°C, washed with a washing solution containing 0.05% Tween 20 in PBS and then blocked with block buffer (PBS containing 0.1% Bovine Serum Albumin) at room temperature for 2 h. Following the washing step with a washing buffer (PBS containing 0.05% Tween 20), serially diluted serum samples were added to the plates. IgG was detected with a Goat Anti-Mouse IgG/HRP (Solarbio, China). ELISA plates were developed with TMB Single-Component Substrate solution (Solarbio) and stop solution (2 M H2SO4). After adding the stop solution, the plates were read at 450 nm optical density (OD450).
10
Protein Expression Analysis of M-DM1 Treated Macrophages
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THP-1 cells differentiated into M0, M1, M2 macrophages, and TAMs using the methods described above were incubat ed with M-DM1 (2 μM) for 24 h. The cells were washed and proteins were extracted using RIPA buffer (Thermo Fisher Scientific) supplemented with 0.1% sodium dodecyl sulfate (SDS) and protease inhibitor. The protein concentrations were measured using the BCA reagent kit (Thermo Fisher Scientific). Proteins were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.
Total protein was extracted from tumor tissues using RIPA buffer and quantified with BCA assay. β-actin mouse polyclonal antibody (sc-1836, 1:2000; Santa Cruz, Dallas, TX, USA), PCNA mouse polyclonal antibody (sc-56, 1:1000; Santa Cruz), caspase-3 Antibody (#9662, 1:1000; Cell Signaling Technology, Danvers, MA, USA), and cleaved-caspase-3 (Asp175) (#9661, 1:1000; Cell Signaling Technology) were used as primary antibodies. Mouse anti-goat IgG-HRP (sc-2354, 1:2000; Santa Cruz), goat anti-rabbit IgG/HRP (SE134, 1:2000; Solarbio), and goat anti-mouse IgG/HRP (SE131, 1:2000; Solarbio) were used as secondary antibodies. The protein bands were detected using a chemiluminescence reagent kit (SurModics, Eden Prairie, MN, USA). The intensity of the protein was normalized to the intensity of β-actin using ImageJ (NIH).
Total protein was extracted from tumor tissues using RIPA buffer and quantified with BCA assay. β-actin mouse polyclonal antibody (sc-1836, 1:2000; Santa Cruz, Dallas, TX, USA), PCNA mouse polyclonal antibody (sc-56, 1:1000; Santa Cruz), caspase-3 Antibody (#9662, 1:1000; Cell Signaling Technology, Danvers, MA, USA), and cleaved-caspase-3 (Asp175) (#9661, 1:1000; Cell Signaling Technology) were used as primary antibodies. Mouse anti-goat IgG-HRP (sc-2354, 1:2000; Santa Cruz), goat anti-rabbit IgG/HRP (SE134, 1:2000; Solarbio), and goat anti-mouse IgG/HRP (SE131, 1:2000; Solarbio) were used as secondary antibodies. The protein bands were detected using a chemiluminescence reagent kit (SurModics, Eden Prairie, MN, USA). The intensity of the protein was normalized to the intensity of β-actin using ImageJ (NIH).
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