Protein extracts were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions on 10% and 12% gels and then transferred to PVDF membranes at 200 mA for 1 h in Tris-glycine buffer containing 20% methanol. Membranes were blocked with 5% skim milk for 2 h and incubated at 4°C overnight with diluted primary antibody against E4F1 (1 : 100), GRP78 (1 : 1000), activating transcription factor 6 (ATF6) (1 : 500), C/EBP homologous protein (CHOP) (1 : 500), B-cell lymphoma-2 (Bcl-2) (1:500), cysteinyl aspartate-specific proteinase 12 (Caspase12) (1 : 1000), and Caspase3 (1 : 100). Following incubation with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (1 : 5000, Santa Cruz Biotechnology, USA), the bands were detected using an ECL reaction. The optical density (OD) was determined using an ImageJ VCD gel imaging system.
Horseradish peroxidase hrp conjugated goat anti rabbit igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG is a reagent used in various immunoassay techniques. It consists of goat-derived antibodies that specifically recognize and bind to rabbit immunoglobulin G (IgG), with horseradish peroxidase enzyme molecules attached to them. The HRP enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated donkey anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Mouse anti α tubulin, by Merck Group (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Abcam (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Rabbit polyclonal anti nek3 antibody, by Abcepta (1 mentions)
Dm il led microscope, by Leica Microsystems (1 mentions)
β tubulin, by Abcam (1 mentions)
Superblock buffer, by Thermo Fisher Scientific (1 mentions)
Bkm120, by Selleck Chemicals (1 mentions)
Byl719, by Selleck Chemicals (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
Bez235, by Selleck Chemicals (1 mentions)
Her3 xp, by Cell Signaling Technology (1 mentions)
P her3 y1197, by Cell Signaling Technology (1 mentions)
E6 2e 3f8, by Euromedex (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
P rps6 s235 236, by Cell Signaling Technology (1 mentions)
26 protocols using horseradish peroxidase hrp conjugated goat anti rabbit igg
1
Western Blot Analysis of ER Stress Markers
2
Western Blotting of IRF-2 Protein
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Western blotting procedure was carried out according to the manufacturer's instructions. Briefly, frozen tissue samples were crushed and ground into fine powder in liquid nitrogen and lysed with 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocked with 5% fat-free dry milk for 1 hour, the membranes were incubated with anti-human IRF-2 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin antibody (1:1000, Epitomics, Burlingame, USA) overnight at 4°C. The next day after washing with TBST buffer, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000, Santa Cruz, CA) for 1 hour, and IRF-2 expression was detected using ECL prime Western blotting detection reagent (Amersham). β-actin acted as a loading control.
3
Immunohistochemistry Evaluation of NEK3 Expression
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Immunohistochemistry was performed in accordance with previous studies.[20 (link),21 (link)] In brief, sample sections were incubated overnight at 4°C with rabbit polyclonal anti-NEK3 antibody (1:200, Abgent), followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:500, Santa Cruz, Bolivia). The sections were counterstained with hematoxylin, and then mounted for observation under the DM IL LED microscope (Leica Microsystems GmbH). The immunostaining results were independently assessed by 2 pathologists who blinded to the clinical data of the patients. The intensity of immunostaining was graded as 0 (no or weak staining), 1 (moderate staining), and 2 (strong staining). At least 5 areas of each section were viewed and the percentage of NEK3+ cells was scored according to the following criteria: 1 (<50% NEK3+ cells), 2 (50–75% NEK3+ cells), and 3 (>75% NEK3+ cells). Then, A semiquantitative histopathology score was obtained by multiplying the staining intensity score with the percentage score. The average of histopathology score was applied as the cut-off to differentiate between low and high expression of NEK3.
4
Comprehensive PI3K Pathway Protein Analysis
5
Notch1 and Jagged1 cDNA Overexpression
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Full-length Notch1 and Jagged1 cDNA lacking the 3′-untranslated region (UTR) were purchased from GeneCopoeia (Rockville, MD, USA) and subcloned into the eukaryotic expression vector pIRES (Clontech Laboratories, Inc., Mountain View, CA, USA). The empty pIRES vector was used as a negative control. Rabbit monoclonal anti-Notch1 (cat no. EP1238Y) and anti-Jagged1 (cat no. EPR4290) were purchased from Epitomics (Abcam, Cambridge, UK), mouse monoclonal anti-GAPDH (cat no. MAB374) was purchased from EMD Millipore (Billerica, MA, USA), and mouse monoclonal anti-vimentin (cat no. sc-6260), anti-fibronectin (cat no. C6F10) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat no. sc-2004) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Synthetic miR-34a mimic and negative control (NC) were purchased from GenePharma Co., Ltd. (Shanghai, China). The miR-34a LNA 5′-digoxigenin (DIG)-labeled (cat no. 612537-340) and scrambled LNA 5′-DIG-labeled probes (cat no. 699004-340) were purchased from Exiqon A/S (Vedbaek, Denmark). The probe sequences are as follows: miR-34a, AGG GCA GTA TAC TTG CTG AT; and scramble, GTG TAA CAC GTC TAT ACG CCC A. The probes were detected using an enhanced sensitive ISH detection kit (cat no. MK1030; Wuhan Boster Biological Technology, Ltd., Wuhan, China).
6
Western Blot Analysis of GFP Protein
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Western blot assay was performed as described by Hinojosa-Moya et al. (2013 (link)). In resume, total protein was extracted by grinding plant tissue in liquid nitrogen, homogenized in protein extraction buffer, and centrifuged at 13,000 g for 2 min. Supernatants were recovered and dissolved in SDS-PAGE sample buffer. Protein concentration was determined using a spectrophotometer (NanoDrop™ 1000; Thermo Scientific, Waltham, MA) and all samples homogenized to the same concentration followed by 12% SDS-PAGE. Total proteins were transferred for 1 h at 100 V to polyvinylidene difluoride (PVDF) membranes (Whatman), blocked for 2 h in blocking solution (PBS 1X, 5% non-fat milk, and 0.1% Tween 20) followed by rinsing with PBS 1X, and incubated overnight at 4°C with the polyclonal GFP antibody (diluted 1:2000 in 1X PBS, 5% non-fat milk, and 0.1% Tween 20). Membranes were washed (1X PBS, 0.1% Tween 20) 5 times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA) at 1:5000 in PBS 1X containing 2.5% skim milk during 2 h. Finally, after several washes, the signal was detected with HRP color development reagent (Amersham Prime Western Blotting Kit Reaction-ECL™-GE Healthcare™) and revealed in an Amersham Hyperfilm (ECL™-GE Healthcare™) film.
7
Western Blot Analysis of Dental Proteins
8
Stem Cell Marker Expression in Pancreatic Cancer
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Total proteins were isolated with lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM DTT, 0.5% Pharmalyte 3–10) supplemented with a protease inhibitor cocktail (all reagents were from Sigma-Aldrich GmbH, Steinheim, Germany). An amount of 10 μg of proteins was resolved on a 10% SDS-PAGE and subsequently transferred onto PVDF membrane (GE Healthcare, Chicago, IL, USA). After blocking with 5% BSA/TBST for 1 h, the membranes were incubated with primary antibodies (OCT-4 1:1000, Nanog 1:500, CD105, Merck KGaA, Darmstadt, Germany) overnight. After incubation, the membranes were washed with TBS-T three times, 10 min each, and again incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:20,000, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. After washing with TBS-T three times, the immunoreactive protein bands were visualized with the ECL kit (Merck KGaA, Darmstadt, Germany). Human β-actin served as the normalizing marker and total lysates from cell line PATU-8988S (pancreatic carcinoma) was used as positive control as cancerogenic cell lines express increasing stem cell markers.
9
Wnt3a, sFRP-3, and Dkk-1 Regulation of Stem Cell Differentiation
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Human Wnt3a recombinant protein (rWnt3a), secreted frizzled-related protein-3 (sFRP-3), and dickkopf-1 (Dkk-1) were purchased from R&D Systems (USA). The recombinant rhTGF-β3 protein was purchased from Peprotech (Rocky Hill, NJ, USA). Fetal bovine serum (FBS) and Eagle's minimal essential medium (MEM) were purchased from HyClone (South Logan, UT, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO). MicroBeads conjugated to monoclonal mouse anti-human CD105 and CD166, anti-mouse secondary antibodies coupled to magnetic beads, and primary antibodies anti-human CD105-PE, anti-human CD166-FITC, anti-mouse CD105, anti-mouse CD166 were obtained from Miltenyi Biotec (Germany), and anti-human β-catenin was obtained from Cell Signaling Technology (USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was provided by Santa Cruz (USA). Trizol and Superscript III kits were purchased from Invitrogen (USA). The RNeasy mini kit was obtained from Qiagen (Netherlands).
10
Membrane Protein Immunoblotting Assay
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Cultured strips of RCA were homogenized and membrane lysate prepared (Liu et al. 2001 ). Equivalent amounts of membrane proteins were run on a 4–15% polyacrylamide gradient gel. After electrophoresis, the proteins were electroblotted to a PVDF membrane. The membrane was subsequently blocked overnight (at 4°C) with 5% non‐fat dry milk dissolved in a Tris‐buffered saline containing 0.1% Tween 20 (TBST). After decanting off the blocking solution, the membrane was incubated in a 1:100 dilution of rabbit polyclonal anti‐α1C (Alomone Labs, Jerusalem, Israel) for 60 min followed by three washes (10 min each) using TBST. The membrane was then incubated with horseradish peroxidase (HRP) conjugated goat anti‐rabbit IgG (1:1000; Santa Cruz Biotech, Santa Cruz, CA) for 60 min and subsequently washed (3x) with TBST. Chemiluminescence permitted detection of the HRP‐labeled antibody using an AlphaImager Documentation and Analysis System (Alpha Innotech Corp., San Leandro, CA). Likewise, band densitometry was analyzed using AlphaImager software. Smooth muscle β‐actin (1:1000; Abcam, Cambridge, MA) was used as a loading control in Western blots.
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