After washing with PBS, harvested cells were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche, Switzerland). Protein concentrations were determined with BCA Protein Assay Kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Equal amounts of proteins were subjected to SDS-PAGE and Western blotting. The primary Abs were as follows, anti-GSTM3 Ab (1:500, Proteintech, USA), anti-GSTP1 Ab (1:500, Proteintech, USA) and anti-GAPDH Ab (1:2000, Proteintech, USA). The second Abs were as follows, goat anti-mouse HRP conjugated IgG (1:4000, Proteintech, USA) and goat anti-rabbit HRP conjugated IgG (1:4000, Proteintech, USA). Western blotting signals were detected and developed using a BIO-RAD ChemiDoc XRS chemiluminescence system (Bio-Rad Inc., CA, USA).
Hrp conjugated goat anti rabbit igg
Manufactured by Proteintech
Sourced in United States, China
HRP-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. The HRP (horseradish peroxidase) conjugate allows for colorimetric or chemiluminescent detection in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg, by Proteintech (22 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Goat anti mouse igg, by Proteintech (5 mentions)
Anti gapdh, by Proteintech (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Anti gapdh antibody, by Proteintech (2 mentions)
Ab8245, by Abcam (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Ab3927, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Immobilon hrp substrate, by Merck Group (2 mentions)
Tanon 4500 gel imaging system, by Shanghai Tanon Science & Technology (2 mentions)
Sa00001 2, by Proteintech (2 mentions)
Ecl reagent, by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
63 protocols using hrp conjugated goat anti rabbit igg
1
Western Blot Analysis of GSTM3 and GSTP1
2
Comprehensive Protein Immunodetection Protocol
3
Comparative Proteomics of Influenza-Infected Cells
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The extracted total cell lysate and mitochondrial proteins of H5N1 and H9N2 virus-infected A549 cells were quantified with a BCA kit (Sangon Biotech, Shanghai, China). Mitochondrial proteins (40 µg) and total cell lysate (40 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (BBI Life Sciences). After blocking with 5% (w/v) skim milk in TBST (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1% [v/v] Tween-20) for 1 h at 37 °C, membranes were incubated separately overnight at 4 °C with rabbit monoclonal or polyclonal antibodies against ECHS1 (ab170108), MDH2 (ab181873) (Abcam), ATP5F1 (15999-1-ap), HSPA1L (13970-1-ap), BAX (50599-2-Ig), and Caspase 3 (66470-2-Ig) (Proteintech). After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (used at a 1:5000 dilution, Proteintech) for 1 h at room temperature and were then washed three times with TBST. The immunoreactive protein bands were detected using enhanced chemiluminescence reagent (ECL; Advansta, CA, USA), with TOM40 (18409-1-ap) (Proteintech) and β-actin (66009-1-Ig) (Proteintech) as the loading controls.
4
Western Blot for UPF3A/UPF3B Proteins
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Cells or mouse tissues were lysed with RIPA buffer (20-188; Sigma-Aldrich) supplemented with combinations of protease/phosphatase inhibitors (APExBIO). Around 40–60 μg protein was separated with gradient SDS–PAGE gel (4–20%, ACE Biotechnology). The following primary antibodies were used: rabbit anti-UPF3A+UPF3B (ab269998, Abcam, 1:1,000); mouse anti-β-actin (A5441; Sigma-Aldrich, 1:10,000); mouse anti-Lamin B1 (sc-374015; Santa Cruz). The secondary antibodies used in these studies were HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:2,000; Proteintech).
5
TRIP13 Immunohistochemical Staining in Mice Xenograft
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Three groups of sections were collected from mice xenograft experiment. Sections were first deparaffinized in xylene after being placed at 60 °C overnight, and then dehydrated with gradient concentrations of ethanol and washed with 3% H2O2. Hot sodium chloride citrate buffer was used to renovate antigen for 20 min. The samples were incubated with TRIP13 antibody (ab204331, 1:100, Abcam, USA) at 4 °C overnight. The samples were washed by PBS and incubated at room temperature for 30 min with secondary antibody HRP-conjugated goat anti-Rabbit Ig G (Protein tech, USA). Diaminobenzidine (DAB) was performed as chromogen and hematoxylin was used to redye. Staining patterns were determined in selected representative slices. Staining intensity was categorized as negative, popcorn, yellow or brown.
6
Western Blot Analysis of Autophagy Markers
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Total protein lysates were extracted from the mice tongue from each experimental group by using a Total protein lysate kit (catalog no. KGP250; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Protein concentration was quantified using the bicinchoninic acid assay (catalog no. KGP902; Nanjing KeyGen Biotech Co., Ltd., and 30 µg of total protein was subjected to 12% SDS-PAGE. The fractionated samples were transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), immersed in 5% blotto at room temperature for 60 min to block non-specific binding and then incubated with anti-GAPDH antibody (1:5,000; catalog no. 10494-1-AP; Proteintech Group), anti-LC3B antibody (1:1,000; catalog no. 14600-1-AP; Proteintech Group), anti-P62 antibody (1:500; catalog no. WL02385; Wanleibio Co., Ltd.) and anti-Beclin 1 antibody (1:500; catalog no. WL02508; Wanleibio Co., Ltd.) overnight at 4°C. The membranes were then incubated with the HRP-conjugated goat anti-rabbit IgG (1:5,000; catalog no. SA00001-2; Proteintech Group) for 1 h at room temperature. The specific bands were detected using the Immobilon Western Chemiluminescent HRP Substrate detection kit (EMD Millipore). The intensities of the protein bands were quantified with Quantity One 4.5.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
7
Western Blot Analysis of Liver Tissues
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The protein extracts from liver tissues and CFSC cells were prepared in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktails (Roche, Shanghai, China). Then, the protein samples were separated using SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Shanghai, China). After blocking with 5% skim milk, the membranes were incubated with mouse anti-α-SMA antibody (Sigma Aldrich, Shanghai, China) or rabbit anti-GAPDH antibody (Proteintech, Wuhan, China) overnight at 4 °C and subsequently incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China) for 2 h at room temperature. Finally, the bands were examined with an ECL assay kit (NCM Biotech, Suzhou, China).
8
Immunohistochemical Analysis of Brain Samples
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For immunohistochemical observation, the sections of the brain sample were deparaffinized, rehydrated, and treated with 3% H2O2, followed by the addition of citrate buffer. The blocked sections were incubated with TXNIP antibody (catalog number: 18243-1-AP, Proteintech, Wuhan, China), or NLRP3 antibody (catalog number: 19771-1-AP, Proteintech), followed by incubation with the HRP-conjugated goat anti-rabbit IgG (catalog number: SA00001-2, Proteintech). The sections were subsequently treated with a 3,3-diaminobenzidine substrate system. The slides were observed using an Olympus light microscope (BX51, Olympus corporation).
9
Protein and Gene Expression Analysis
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Total protein was isolated from treated cells. Protein concentrations were measured using a BCA protein assay kit (TransGen Biotech, Beijing, China). Western blot for validation of Mx was performed as previous described (Wang et al., 2022 ). Rabbit anti-chicken Mx (Proteintech, Wuhan, China) was used as the primary antibody, and HRP conjugated goat anti-rabbit IgG (Proteintech) as the second antibody. Meanwhile, β-actin as reference was measured using mouse anti-β-actin monoclonal antibody (Proteintech) as the primary antibody, and HRP conjugated goat anti-mouse IgG (Proteintech) was utilized as secondary antibody.
Total RNA was extracted from treated cells via Trizol reagent (Accurate Biotechnology, Hunan, China). RT-qPCR for chicken IRF-7, IFN-α, IFN-β, TNF-α, p21, p27, and Bak mRNA validation was performed as previous described (Xue et al., 2022 (link)). Meanwhile chicken β-actin was taken as the reference gene. The 2−ΔΔCt algorithm was employed to estimate the relative expression level of genes. The information of primers was listed inTable S3 .
Total RNA was extracted from treated cells via Trizol reagent (Accurate Biotechnology, Hunan, China). RT-qPCR for chicken IRF-7, IFN-α, IFN-β, TNF-α, p21, p27, and Bak mRNA validation was performed as previous described (Xue et al., 2022 (link)). Meanwhile chicken β-actin was taken as the reference gene. The 2−ΔΔCt algorithm was employed to estimate the relative expression level of genes. The information of primers was listed in
10
Western Blot Analysis of Myocardial Proteins
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The Western blot was conducted as previously described (Zhang et al., 2019 (link)). Total protein was extracted from myocardial tissues or cells by RIPA buffer containing protease and phosphatase inhibitor cocktail. Protein concentration was determined using Bradford assay. Equal amounts of proteins from each sample were subjected to 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, blots were incubated with the following antibodies at 4°C overnight: ACSL4 (Abclonal, A14439), GPX4 (Abclonal, A1933), FTH1(CST, 4393), TfR1 (Abclonal, A5865), NCOA4 (Abclonal, A5695), and LC3 A/B (CST, 12,741). After three washes with TBST, these membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Proteintech; SA00001–2). Immune complexes were visualized with enhanced chemiluminescence. β-tubulin (ProteinTech; 10068-1-AP) was used as the endogenous control. The optical density of bands was analyzed by ImageJ software.
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