Cells were disrupted using RIPA lysis buffer and cleared by centrifugation. Proteins were resolved on a 12% SDS-PAGE gel, transferred to a PVDF membrane (Millipore, Etobicoke, ON, Canada), which was blocked with 5% non-fat milk powder in PBS: 0.1% Tween 20 (PBST), then incubated overnight with the appropriate primary antibody at 4°C. Primary antibodies used were rabbit polyclonal anti-Cdx2 (1/1000 dilution, Savory et al., 2009); anti-NICD (1/500 dilution, BD Biosciences); anti-β-actin (1/2000 dilution, Abcam, Branford, CT, USA) or anti-CyclophilinB (1/10,000 dilution, Abcam). Membranes were then washed, incubated with secondary antibodies (HRP-conjugated anti-rabbit or anti-mouse IgG, as appropriate; 1/25,000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) and immunoreactivity detected by ECL (Millipore) according to the manufacturer’s instructions.
Hrp conjugated anti mouse or anti rabbit igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The HRP-conjugated anti-mouse or anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It is conjugated with the enzyme Horseradish Peroxidase (HRP), which catalyzes a colorimetric reaction, enabling the detection and quantification of the target antigen. This product can be used to detect and measure mouse or rabbit primary antibodies in applications such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (4 mentions)
Ecl prime western blotting detection system, by GE Healthcare (4 mentions)
Las 3000, by Fujifilm (4 mentions)
Complete mini, by Roche (4 mentions)
β actin, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Anti cyclophilin b, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Anti β actin, by Abcam (1 mentions)
Ab51049, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
A5316, by Merck Group (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Anti β actin antibody clone ac 74, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
11 protocols using hrp conjugated anti mouse or anti rabbit igg
1
Western Blot Analysis of Cdx2, NICD, and Actin
2
Westerns Blotting Assay for Wnt Signaling
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Whole cell lysates were extracted using RIPA buffer supplemented with protease inhibitor cocktail. Protein lysate were separated by SDS-polyacrylaminde gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Millipore, MA, USA) for western blotting analyses. Primary antibodies against Cripto-1 (1:1000, ab108391, Abcam), total β-catenin (1:2500, 610153, BD Transduction Laboratories, San Jose, CA), Dishevelled-3 (DVL3) (1:1000, sc-8027, Santa Cruz Biotechnology), Frizzled-7 (FZD7) (1:1000, ab51049, Abcam), LRP6 (1:1000, #2560, Cell Signaling) and β-actin (1:80,000, A5316, Sigma-Aldrich, St. Louis, MO, USA) were incubated at 4 °C overnight. HRP-conjugated antirabbit or antimouse IgG were used as secondary antibodies at 1:5000 where appropriate (Santa Cruz Biotechnology, Texas, USA). The signals were visualized using the enhanced chemiluminescence method.
3
Immunoblotting Analysis of Receptor Tyrosine Kinase Signaling
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Immunoblotting was performed as described previously [80 (link)]. Briefly, 40 μg of each cell lysate was separated by 10% SDS–PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 5% skimmed milk for 1 h and incubated overnight at 4 °C with primary antibodies against pERBB1 (D7A5:1:1000), CST; ERBB1 (D38B1; 1:1000), CST; p-c-MET (D26; 1:1000), CST; c-MET (4560; 1:1000), CST; pERBB2 (2247s; 1:1000), CST; ERBB2 (2242; 1:1000), CST; pERK (9102; 1:1000), CST; ERK (9101; 1:1000), CST; pAKT (9271; 1:1000), CST; AKT (4691; 1:1000), CST. All these antibodies were purchased from Cell Signaling technologies, Beverly, MA, USA, unless, mentioned specifically. Following washes with PBST, the membranes were incubation with HRP-conjugated anti-rabbit or anti-mouse IgG (1:3000; Santa Cruz Biotechnology, Dallas, Texas, USA) for 1 h. The proteins were visualized using ECL-plus reagents (Amersham Biosciences Corp., USA).
4
Western Blot Analysis of IL18RAP Expression
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Whole-cell lysate was obtained by lysing neutrophils with lysis buffer (Cell Signaling Technology, Danvers, MA, USA) of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 mM PMSF (Sigma-Aldrich, Steinheim, Germany) and protease inhibitor cocktail. Protein from each sample (30 μg) was separated by SDS-PAGE and transferred to PVDF membrane (GE Healthcare Life Science, Freiburg, Germany), followed by incubation with anti-IL18RAP monoclonal antibody (clone 290, Invitrogen, Rockford, IL, USA) or with anti-β-actin antibody (clone AC-74, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C. After incubation with HRP-conjugated antirabbit or antimouse IgG (Santa Cruz, Dallas, TX, USA), membranes were exposed to enhanced chemiluminescence substrate. Signals were detected by a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry reading of the blots was analysed by Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA) and IL18RAP signal intensity was normalized with the β-actin level in the corresponding sample.
5
Western Blot Analysis of Receptor Signaling
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The total cell lysate was extracted with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma‐Aldrich) and Complete Mini (Roche, Basel, Switzerland). Western blot analysis was carried out using the conventional method using the following primary antibodies: anti‐EGFR, phospho‐ (p‐) EGFR (Tyr1068), HER2, p‐HER2 (Tyr1221⁄1222), MET, p‐MET (Tyr1234⁄1235), AKT, p‐AKT (Ser473), p44⁄p42 MAPK, p‐p44⁄p42 MAPK, E‐cadherin, N‐cadherin, vimentin, ALDH1 (Cell Signaling Technology, Danvers, MA, USA) and b‐actin (used as the loading control) (Merck Millipore, Billerica, MA, USA). The secondary antibody was HRP‐conjugated anti‐mouse or anti‐rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS‐3000 (Fujifilm, Tokyo, Japan).
6
Antibodies for Cell Signaling Analysis
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The specific antibody against ARHGEF11exon38(+) isoform was raised using a peptide coded by exon38, SKVVPALPESGQSEP, as an antigen. Mouse mAbs against RhoA, Rac1, CDC42, E-cadherin, α-catenin, β-catenin, and p27 were obtained from BD Biosciences; rabbit Abs against p-EGFR, EGFR, p-Erk1/2, Erk1/2, p-p38, p38, cyclin-D1, cyclin-E1, cyclin-A2, cyclin-B1, ARHGEF2, keratin8/18, and vimentin were from Cell signaling; rabbit pAb against N-cadherin was from EMD; rabbit Abs against ARHGEF18, Gα13, p15, and Ki67 were from Genetex; rabbit Abs against ZO-1, ZO-2, ZO-3, occludin, claudin-3, and claudin-4 were from Invitrogen; mouse mAbs against ARHGEF11, Bcl-2, and Bcl-xL were from Santa Cruz Biotech.; mouse mAb against vinculin was from Sigma; mouse mAbs against GAPDH, GFP, and Myc were from Wako; Alexa Fluor 488/594-conjugated anti-mouse or anti-rabbit IgG, and Alexa Fluor 594-conjugated phalloidin were from Invitrogen; HRP-conjugated anti-mouse or anti-rabbit IgG were from Santa Cruz Biotech.
7
Western Blot Analysis of Signaling Proteins
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Cells were harvested and lysed in a buffer (25 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktails) before being centrifuged at 15,000 rpm for 20 min. The total proteins (20 μg) were loaded into a sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred to PVDF membrane. The membrane was probed with the primary antibody for PPARγ, cyclin D1, cyclin B1, cdk-6, Akt, phospho-Akt, FOXO1, phospho-FOXO1 (Ser 256) (Cell Signaling Technology, Danvers, MA, USA), C/EBPα, p21 (Santa Cruz, Dallas, TX, USA), or cdk-4 (Abcam, Cambridge, UK). The membranes were then incubated with HRP-conjugated antimouse or antirabbit IgG (Santa Cruz) secondary antibodies. The protein levels were visualized with an Enhanced Chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) and quantified using a Fusion Solo system (Vilber Lourmat, Collegien, France). β-actin (Sigma, St. Louis, MO, USA) served as a loading control.
8
Western Blot Analysis of Signaling Proteins
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The total cell lysate was extracted with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma‐Aldrich, St. Louis, MO, USA), and Complete Mini (Roche, Basel, Switzerland). Western blot analysis was carried out by conventional methods using the following primary antibodies: anti‐EGFR, phospho‐ (p‐)EGFR (Tyr1068), HER2, p‐HER2 (Tyr1221/1222), Akt, p‐Akt (Ser473), p44/p42 MAPK, p‐p44/p42 MAPK, cleaved PARP (Asp214) (Cell Signaling Technology, Danvers, MA, USA), and β‐actin (used as loading control) (Merck Millipore, Billerica, MA, USA). The secondary antibody was HRP‐conjugated anti‐mouse or anti‐rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS‐3000 (Fujifilm, Tokyo, Japan).
9
Western Blot Analysis of EMT Markers
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Cells were treated with ethanol (Wako, Osaka, Japan) as a control, 2 ng/mL TGFβ and 100 nM monensin. The total cell lysate was extracted with lysis buffer, a mixture of RIPA buffer, phosphate inhibitor cocktails 2 and 3 (Sigma-Aldrich, St.
Louis, MO, USA) and complete Mini Protease Inhibitor Cocktail (Roche, Switzerland). Western blot analysis was performed by using the following primary antibodies: E-cadherin, vimentin, poly ADP-ribose polymerase (PARP), GAPDH (Cell Signaling Technology, Danvers, MA, USA) and ZEB1 (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibody was HRP-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, the membrane was examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS-3000 (Fujifilm, Tokyo, Japan). The relative band intensities were quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
Louis, MO, USA) and complete Mini Protease Inhibitor Cocktail (Roche, Switzerland). Western blot analysis was performed by using the following primary antibodies: E-cadherin, vimentin, poly ADP-ribose polymerase (PARP), GAPDH (Cell Signaling Technology, Danvers, MA, USA) and ZEB1 (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibody was HRP-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, the membrane was examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS-3000 (Fujifilm, Tokyo, Japan). The relative band intensities were quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
10
Western Blot Analysis of Signaling Proteins
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Cells were washed in ice‐cold PBS and lysed with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma‐Aldrich) and Complete Mini (Roche). The cell lysate was collected after centrifugation at 20 000 g for 15 minutes at 4°C. Protein was quantified using the DC Protein Assay Kit (Bio‐Rad Laboratories), fractionated on SDS‐PAGE and blotted onto a membrane using the Trans‐Blot Turbo Transfer System (Bio‐Rad Laboratories). After the membrane was blocked with 5% skim milk in TBS‐containing 0.05% Tween 20 (T‐TBS) for 1 hour, it was probed with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody for 1 hour at 25°C. Proteins were detected using ECL Prime Western Blotting Detection Reagent (General Electric Company) and by scanning the membrane using ImageQuant LAS 4000 (General Electric Company). The primary antibodies used were as follows: phospho‐HER2‐Tyr877, HER2, phospho‐AKT‐Ser473, AKT, phospho‐MAPK‐Tyr202/204, MAPK, phospho‐SRC family‐Tyr416, YES1, cleaved PARP (Cell Signaling Technology) and Actin (used as the loading control) (Merck Millipore). The secondary antibody was HRP‐conjugated anti–mouse or anti–rabbit IgG (Santa Cruz Biotechnology).
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