After different treatments of the cells and extraction of lysate proteins, the protein concentration was measured using the bicinchoninic acid protein assay (Thermo Fisher Scientific). The equal amounts of proteins (25 μg) were loaded onto 15% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). After membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 for 1 hour, immunoblotting was conducted by incubating with LC3 and β-actin rabbit antibodies (1:1,000) (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The membranes were then washed with tris-buffered saline containing 0.05% Tween-20 and incubated with a horseradish peroxidase-conjugated antirabbit IgG secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. After being washed three times with tris-buffered saline containing 0.05% Tween-20, the proteins bound with the antibody were detected using the ECL chemiluminescence reagent (Thermo Fisher Scientific). We used the Image Lab™ Software (Bio-Rad Laboratories Inc., Hercules, CA, USA) to perform the densitometric analysis of the Western blot results.
Horseradish peroxidase conjugated anti rabbit igg secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody is a laboratory reagent used in immunoassays and Western blotting applications. It consists of an anti-rabbit IgG antibody conjugated to the enzyme horseradish peroxidase, which can catalyze a colorimetric or chemiluminescent reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Anti pro caspase 3, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Standard bicinchoninic acid assay, by Beyotime (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Ecl chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Cd11b, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Gapdh 14c10, by Cell Signaling Technology (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Iblot gel transfer stack, by Thermo Fisher Scientific (1 mentions)
Aggrecan, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Roche (1 mentions)
Cxcl12, by Cell Signaling Technology (1 mentions)
Type 1 collagen, by Abcam (1 mentions)
27 protocols using horseradish peroxidase conjugated anti rabbit igg secondary antibody
1
Western Blot Analysis of LC3 Protein
2
Immunohistochemical Analysis of Tumor-Infiltrating Immune Cells
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Subcutaneous tumors were harvested from mice after the completion of treatment with TKI and/or ICI as described above. Immunohistochemical staining of tumor specimens was performed as reported previously24 . Briefly, sections from formaldehyde-fixed, paraffin-embedded tissue were deparaffinized with xylene and rehydrated in a graded series of alcohol and distilled water. After endogenous peroxidase was blocked with 3% hydrogen peroxide in distilled water, sections were stained with antibodies against mouse CD3, CD8 (Cell Signaling Technology), and CD11b (Abcam) at 4 °C overnight. Sections were then reacted with horseradish peroxidase-conjugated anti-Rabbit IgG secondary antibody (Cell-signaling Technology) for 30 min at room temperature. After incubation in avidin–biotin peroxidase complex for 30 min at room temperature, samples were exposed to diaminobenzidine tetrahydrochloride solution and counterstained using hematoxylin.
Quantitative assessments of the findings of immunohistochemical staining were performed using Fiji software as previously described25 (link). Five sections per tumor were prepared in the immediate vicinity of each tissue, and the number of positive cells after immunohistochemical staining were quantified at five random locations per section.
Quantitative assessments of the findings of immunohistochemical staining were performed using Fiji software as previously described25 (link). Five sections per tumor were prepared in the immediate vicinity of each tissue, and the number of positive cells after immunohistochemical staining were quantified at five random locations per section.
3
Molecular Pathways Regulation Assay
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Almost chemicals and reagents were purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan) unless otherwise noted. Antibodies, phosphorylated-LIM kinase (LIMK)1, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Phosphorylated-Slingshot was purchased from ECM Biosciences (Versailles, KY, USA). Alpha-Fodrin was purchased from Biomol (Plymouth Meeting, PA, USA).
4
Lovastatin Enhances Adenoviral Infection
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CWR22rv, C4-2 and PZ-HPV-7 cells were seeded onto 12-well plates (2.5 × 105 cells per well), and treated with 10 μM lovastatin for 16 hours before infection with AdE4 at 100 vp/cell. DMSO-treated cells were used as the control. Protein preparations (40 μg) were subjected to SDS-PAGE separation, and electroblotted to a nitrocellulose membrane. Antibodies against human CAR, integrin αυ, DR4, DR5 and caspase 3 were purchased from Santa Cruz Biotechnology. Primary antibodies were detected using horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Cell Signaling, Danvers, MA, USA).
5
EGFP+ B Cell Protein Analysis
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EGFP+ transduced splenic B cells were sorted and analyzed by Western blotting as previously described (Jeelall et al., 2012 (link)). Myd88 and phospho-p65 NF-κB primary antibodies obtained from Cell Signaling Technology were used at 1:1,000 dilutions. Horseradish peroxidase–conjugated anti–rabbit IgG secondary antibody purchased from Cell Signaling Technology was used at 1:2,500 dilution. Membranes were reprobed with antibody to αβ tubulin obtained from Cell Signaling Technology at 1:5,000 dilution as a loading control.
6
Western Blot Analysis of Phosphoproteins
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For WB analysis, tissues were lysed and resolved with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The following primary antibodies were used: p-Akt (Ser473) (D9E), p-Akt (Thr308) (D25E6), phosphorylated ribosomal protein S6 kinase beta-1 (p-p70S6K; Thr421/Ser424), and GAPDH (14C10) (1:1000) (all Cell Signaling Technology, Boston, MA, USA). A horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:5000) was used (Cell Signaling Technology, Boston, MA, USA). The results were normalized to the reference protein GAPDH. The analysis was carried out using Image Lab 5.2.1 software (Bio-Rad Inc., Hercules, CA, USA).
7
Immunoblotting of Ezrin Phosphorylation
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Total cell extracts were prepared from PBMCs and EBV-B cells, either untransfected or transfected by nucleofection with pcDNA3.1-ezrin or pcDNA3.1 (mock). Equal amounts of protein from each sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto iBlot Gel Transfer Stacks (Thermo Fisher Scientific). These nitrocellulose membranes were then probed with rabbit polyclonal antibodies directed against ezrin (PA5-86597, Thermo Fisher Scientific), mouse monoclonal antibodies directed against Tyr146-phosphorylated ezrin (sc-166858, Santa Cruz Biotechnology, Santa Cruz, Calif), or rabbit polyclonal antibodies directed against Tyr353-phosphorylated ezrin (3144, Cell Signaling Technology, Danvers, Mass), followed by a horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technology). Membranes were stripped and reprobed with an antibody against glyceraldehyde-3-phosphate dehydrogenase (aka GADPH) (Abcam, Cambridge, United Kingdom) to control for protein loading. Antibody binding was detected by enhanced chemiluminescence (NZYTech, Lisbon, Portugal).
8
Western Blot Analysis of Cell Markers
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CHON-001 cell lysates were washed with PBS and homogenized in radioimmunoprecipitation assay buffer (Roche Diagnostics, Basel, Switzerland). Proteins (30 µg per lane) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were blocked with 5% milk in Tris buffered saline with 0.1% Tween-20 for 2 h at room temperature. Next, they were incubated with primary antibodies against β-actin (cat no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CXCL12 (cat no. 3530; 1:1,000; Cell Signaling Technology, Inc.), type I collagen (cat no. ab34710; 1:1,000; Abcam) and aggrecan (cat no. ab36861; 1:1,000; Abcam) at 4°C overnight. Membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Bands were visualized by SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). Band density was normalized to β-actin.
9
Western Blot Quantification of Phospho-Darpp-32
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Proteins were separated by 10% SDS/PAGE and transferred onto a nitrocellulose membrane. Blocking and incubation with primary and secondary antibodies were performed in 5% (w/v) nonfat dry milk. Primary antibody against phospho‐Darpp‐32/t‐Darpp (T39) (#2301) was from Cell Signaling Technology (Danvers, MA, USA). H62 (sc‐11365), an antibody that recognizes both Darpp‐32 and t‐Darpp, was from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase‐conjugated anti‐rabbit IgG secondary antibody was purchased from Cell Signaling Technology. Chemiluminescence was produced using the Amersham ECL Plus kit (GE Healthcare Bio‐Sciences, Pittsburgh, PA, USA) and captured on X‐ray film.
10
Protein Expression Analysis by Western Blot
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Proteins from cells or blood samples were extracted using the RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was quantified using Bicinchoninic Acid Protein Assay kit. Proteins (30 µg/lane) were separated by using SDS-PAGE on a 12% gel, and then transferred onto the PVDF membranes. Membranes were then blocked with 5% skim milk at room temperature for 1.5 h and incubated with primary antibodies overnight at 4°C: Bcl-2 (cat no. 4223; 1:1,000; Cell Signaling Technology, Inc.), Bax (cat no. 5023; 1:1,000; Cell Signaling Technology, Inc.), and β-actin (cat no. 4970; 1:1,000; Cell Signaling Technology, Inc.). The membranes were finally incubated with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat no. 7074; dilution ratio: 1:5,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. To visualize the protein bands, the ECL detection system (Thermo Fisher Scientific, Inc.) was used. Protein bands were quantified using. β-actin was used as a reference protein for each experiment and each experiment was repeated three times. Densitometric analyzes were performed using ImageJ software (version 1.38X; National Institutes of Health).
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