The EGFR and HER3 protein expression were determined by SDS-PAGE using the Invitrogen XCell SureLock system. Briefly, proteins were extracted from cell pellets with RIPA buffer and protease and phosphatase inhibitors (HALT, ThermoFisher). Fifteen micrograms of protein were resolved by 4–12% SDS-PAGE and transferred to membrane. After blocking with 5% milk in TBS-0.1% Tween-20, membrane was incubated with anti-EGFR-XP (D38B1, Cell Signaling), anti-HER3-XP (D22C5, Cell Signaling), or anti-β-Actin (mAbcam 8226, Abcam) antibody overnight at 4 °C. After incubation with secondary antibody (anti-mouse HRP or anti-rabbit HRP, Amersham) the membrane was visualized by Ammersham ECL (GE Life sciences) and read using a ChemiDoc imaging system (Bio-Rad) and analyzed using image lab touch software 2.2 (Bio-Rad).
Anti mouse hrp
Manufactured by Cytiva
Sourced in United States
Anti-mouse HRP is a secondary antibody conjugated with horseradish peroxidase (HRP) that binds to mouse primary antibodies. It can be used for detection in immunoassays and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit hrp, by Cytiva (9 mentions)
Cell signaling lysis buffer, by Cell Signaling Technology (3 mentions)
Novex 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Western lightning chemiluminescence reagent, by PerkinElmer (3 mentions)
Nonfat dried milk powder, by AppliChem (2 mentions)
Immobilon p transfer membrane, by Merck Group (2 mentions)
Anti flag hrp, by Merck Group (2 mentions)
Shmt2, by Cell Signaling Technology (2 mentions)
Vinculin, by Cell Signaling Technology (2 mentions)
Shmt1, by Cell Signaling Technology (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Vinculin, by Abcam (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Xcell surelock system, by Thermo Fisher Scientific (1 mentions)
Anti β actin mabcam 8226, by Abcam (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Rpn2106, by GE Healthcare (1 mentions)
17 protocols using anti mouse hrp
1
Quantifying EGFR and HER3 Protein Levels
2
Western Blot Analysis of HuH-7 Cells
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HuH-7 and HuH-7-CORE cells were harvested and dissolved in RIPA buffer (Tris-HCl 50 mM, NaCl 150 mM, 1 % sodium desoxycholate, 1 % Triton X-100, 0.1 % SDS, pH 7.5) or in pRb2/p130 buffer as previously described [43 (link)] supplemented with 1X proteinase inhibitor cocktail (Sigma Aldrich, Milan, Italy). Cell lysates (25 μg) were loaded onto a 10 % SDS-PAGE and analyzed under reduced conditions. Electrophoretically separated proteins were transferred on a PVDF membrane and probed with specific monoclonal antibody for 1.5 h at 37 °C or overnight at 4 °C. After extensive washing with TBS-T (Tris-HCl 20 mM, NaCl 137 mM, 0.5 % Tween, pH 7.6), membranes were incubated with appropriate HRP-conjugated secondary antibody. Protein bands were revealed by chemiluminescent substrates (ECL, Amersham Biosciences, Sweden). Antibodies: anti-HCV core protein mouse monoclonal antibody (Anogen Yes Biotech Laboratories. Ltd, Mississauga, Ontario, Canada) A1/3D1clone; anti-pRb2/p130 clone (BD Transduction Laboratories, Franklin Lakes, NJ); anti-pRb H-125 clone (Santa-Cruz Biotechnology, Santa Cruz, CA)¸ anti-p107 C-18 clone (Santa-Cruz); secondary antibody anti-mouse-HRP (Amersham Biosciences, Piscataway, NJ).
3
Analysis of E2F and HIF-1α Proteins
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Whole-cell lysates were prepared using a lysis buffer containing 150 mM NaCl, 1.0% NP40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris (pH 8.0), supplemented with Protease Inhibitor Cocktail (Roche). Protein lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Membranes were probed with the following antibodies: E2F7 (Santa Cruz, sc-66870), E2F8 (Abnova, H00079733-M01; Abcam AB109596), HIF1α (BD Biosciences, 610959), HDAC1 (sc-7872), E2F1 (sc-193), Mouse IgG HRP-linked whole Ab (GE Healthcare, NA931), Rabbit IgG HRP-linked whole Ab (GE Healthcare, NA934). As secondary antibodies, anti-rabbit-HRP (Amersham Biosciences, NA934; 1:5000) and anti-mouse-HRP (Amersham Biosciences, NA931; 1:5000) were used. All antibodies were diluted in 4% non-fat milk in TBST. Immuno-probed blots were subjected to standard ECL reagents as described by the manufacturer (GE Healthcare, RPN2106).
4
Protein Extraction and Western Blotting
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Whole cell lysates were made in RIPA buffer (150 mM NaCl, 1% IgeCal-CA 360, 0.1% SDS, 50 mM Tris, pH-8.0, 0.5% Sodium deoxycholate). Cytoplasmic and nuclear proteins were extracted as per the manufacturer’s instructions (Thermo Scientific). Lysates were resolved on an SDS-PAGE gel. Protein was a transferred on a nitro-cellulose membrane and blocked in Blotto (5% milk powder, 0.2% Tween-20 in PBS). Membranes were probed over-night with primary antibody. Membranes were washed and incubated with horse-radish peroxidase (HRP) labeled secondary antibody. Membranes were developed using ECL reagents (Amersham). Primary Antibodies: anti-RBPSUH, anti-phospho Zap 70 (Y319), anti-Zap70, anti-HDAC (Cell signaling), anti-c-Rel (Santa Cruz Biotechnology) anti-Actin (Sigma), anti-c-Myc (9E10) was obtained from Dr. Dominique Alfandari. Secondary Antibody: anti-Rabbit-HRP, anti-Mouse-HRP (Amersham).
5
Western Blot Analysis of Protein Expression
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Cells were lysed in 2x sample buffer (0.125M Tris pH 6.8; 4% SDS; 20% glycin; 5% β-mercaptoethanol; 0.025% bromphenol blue). Proteins were separated by SDS-PAGE and transferred onto Immobilon-P Transfer membranes (Millipore) by wet tank or SemiDry blotting. Membranes were blocked in 5% nonfat dried milk powder (AppliChem) in PBS/0.1% Tween20 and subsequently incubated in 5% nonfat dried milk powder containing primary antibodies. Used primary antibodies: anti-Flag HRP (mouse monoclonal; clone M2; Sigma; 1:10 000), anti-b-Tubulin I (Sigma; 1:500 000/1:1 000 000). Used secondary antibodies: anti-mouse HRP (Amersham; 1:10 000). For HRP detection ECL Western Blotting Detection Reagent (Amersham) and WesternBright Quantum (advansta) were used with Luminiscent Image Analyser LAS4000 (Fujifilm). ImageJ was used to quantify signals.
6
Western Blot Analysis of Notch Ligands
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Cells/embryos were lysed in 2x sample buffer (0.125 M Tris pH 6.8/4% SDS/20% glycin/5% beta-mercaptoethanol/0.025% bromphenol blue). Proteins were separated by SDS-PAGE and transferred to Immobilon-P Transfer membranes (Millipore) by wet tank blotting. Blots were blocked in 5% nonfat dried milk powder (AppliChem) in PBS/0.1% Tween 20. Primary antibodies: anti-HA HRP (rat monoclonal; clone 3F10, Roche; HRP, horseradish peroxidase-conjugated; 1:5,000–1:10,000), anti-Flag HRP (mouse monoclonal; clone M2, Sigma), anti-GFP HRP (mouse monoclonal; MACS molecular; 1:10,000), anti-DLL1 (1F9, rat monoclonal, [21 (link)] 1:1,000), anti-DLL4 (rabbit polyclonal against peptide C-GKIWRTDEQNDTLT; BioGenes; 1:50–1:100), anti-β-actin (mouse monoclonal; MP Biomedicals; 1:250,000–1:500,000), anti-β-tubulin I (Sigma; 1:500,000). Secondary antibodies: anti-mouse HRP, anti-rat HRP, anti-rabbit HRP (Amersham; 1:10,000). HRP was detected with ECL Western Blotting Detection Reagents (Amersham) with the Luminescent Image Analyser LAS-4000 (Fujifilm); signals were quantitated with ImageJ software.
7
Western Blot Analysis of SHMT Proteins
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Cells were lysed in Cell Signaling Lysis Buffer (Cell Signaling Technology) [13 (link)], resolved by gel electrophoresis using Novex 4–12% Bis-Tris Gels (Invitrogen), transferred to a nitrocellulose membrane (Bio-Rad), and blocked for 1 h in 5% BSA (Sigma). Blots were incubated in primary antibody to SHMT1 (Cell Signaling, #80715), SHMT2 (Cell Signaling, #12762) or Vinculin (Cell Signaling, #13901), followed by the secondary antibodies anti-rabbit HRP (Amersham) or anti-mouse HRP (Amersham). Bound antibody was detected using the Western Lightning Chemiluminescence Reagent (Perkin Elmer).
8
Quantifying NOTCH1 Signaling and Expression
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Western blots were stained with antibodies specific for γ-secretase–cleaved NOTCH1 (Val1744; #4147 or #2421; Cell Signaling Technology), the intracellular transcriptional activation domain of NOTCH1 (Hasserjian et al., 1996 (link)), or the C terminus of NOTCH1 (#SC-6014 [C-20]; Santa Cruz Biotechnology). Control stains were performed with antibodies specific for actin (#ACTN05; Thermo Fisher Scientific), vinculin (#2907; Abcam), or GAPDH (#137179; Santa Cruz Biotechnology). Blots were developed with anti-rabbit HRP (#NA9340V; Amersham) or anti-mouse-HRP (#NA9310V; Amersham).
Expression of FR1 and FR2 was determined using isoform-specific antibodies (#MAB5646, R&D Systems; #103988, Abcam). Immunofluorescence staining was performed on permeabilized cells using a murine monoclonal antibody against NOTCH1 (3294, Abcam), and species-specific secondary antibodies linked to Alexa Fluor 488. Slides were mounted with Prolong Gold anti-fade reagents and counterstained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal microscope at 100× power. Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described (Roti et al., 2013 (link)).
Expression of FR1 and FR2 was determined using isoform-specific antibodies (#MAB5646, R&D Systems; #103988, Abcam). Immunofluorescence staining was performed on permeabilized cells using a murine monoclonal antibody against NOTCH1 (3294, Abcam), and species-specific secondary antibodies linked to Alexa Fluor 488. Slides were mounted with Prolong Gold anti-fade reagents and counterstained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal microscope at 100× power. Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described (Roti et al., 2013 (link)).
9
Immunoblot Analysis of Signaling Proteins
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Cells and liver tissue were homogenized using E1A lysis buffer (50 mM HEPES pH7.6; 250 mM NaCl; 5 mM EDTA; 0.5% NP40). Protein lysates were prepared from liver samples, and 40 μg lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose and analysed by immunoblotting. Membranes were probed with the following antibodies: anti-A20 (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase-3 (Cell Signaling Technology), JNK (Santa Cruz Biotechnologies), phospho-JNK (Cell Signaling Technology), IκBα (Santa Cruz Biotechnologies), phospho-IκBα (Cell Signaling Technology), Erk (Cell Signaling Technology), phospho-Erk (Cell Signaling Technology) and anti-actin (Santa Cruz Biotechnologies). As secondary antibodies, anti-rabbit-HRP, anti-mouse-HRP and anti-goat-HRP were used (Amersham Bioscience, Buckinghamshire, UK).
10
Immunoblotting for SHMT1 and SHMT2
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