Western blot analysis was performed as described previously45 (link). Samples were resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore) using a semi-dry transfer cell (Bio-Rad). The membranes were blocked with a blocking buffer (total 0.1% Tween 20 and 4% skim milk in PBS) and incubated with primary antibodies in the blocking buffer overnight at 4 °C. The membranes were washed three times for 10 min with 0.1% Tween 20 in TBS, and incubated with secondary antibodies in the blocking buffer for 2 h at room temperature. Signals were detected using Chemi-Lumi One L (Nacalai, Japan) or Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific). We used a rat monoclonal anti-HA (1:5,000, Roche, 3F10) antibody as the primary antibody and horseradish peroxidase-conjugated anti-mouse IgG (1:10,000, Zymed) antibody as the secondary antibody.
Horseradish peroxidase conjugated anti mouse igg
Manufactured by Zymo Research
Horseradish peroxidase–conjugated anti-mouse IgG is a laboratory reagent used for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassays. It consists of anti-mouse IgG antibodies 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
Chemi lumi one l, by Nacalai Tesque (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Pierce western blotting substrate plus, by Thermo Fisher Scientific (1 mentions)
Semi dry transfer cell, by Bio-Rad (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Siah1, by Abcam (1 mentions)
Las 3000 mini system, by Fujifilm (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
4 protocols using horseradish peroxidase conjugated anti mouse igg
1
Western Blot Immunodetection Protocol
2
Western Blot Analysis of FLAG and GFP Proteins
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Western blot analysis was performed as previously described (39 ). Briefly, HEK293T cells were washed with Tris-buffered saline (TBS) twice and lysed in an SDS-sample buffer. The samples were resolved by SDS-PAGE and transferred to PVDF membrane using the iBlot system (Invitrogen). The membranes were blocked with blocking buffer (3% skim milk and 0.05% Tween 20 in TBS) and incubated with primary antibodies overnight at 4 °C. The membranes were washed with 0.05% Tween 20 in TBS three times for 10 min each and then incubated with secondary antibodies for 2 h at room temperature. Signals were detected using Chemi-Lumi One L (Nakalai Tesque). We used the following primary antibodies: mouse anti-FLAG M2 (1:10,000, Sigma, F1804) and rabbit anti-GFP (1:2500, MBL, 598). The following secondary antibodies were used: horseradish peroxidase–conjugated anti-mouse IgG (1:10,000, Zymed) and anti-rabbit IgG (1:10,000, Jackson Laboratory).
3
Western Blot Analysis of Breast Cancer Cell Proteins
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Proteins obtained from breast tumors or MDA-MB-231 and MCF-7 cells transfected with miR-944 precursor (50 nM) or scramble as described above, were separated on 10 % polyacrylamide gels and transferred to PVDF membrane (Millipore). Membrane was incubated overnight at 4 °C with α-actinin-1 (sc-17829, Santa Cruz Biotechnology) or SIAH1 (ab2237 Abcam) primary antibodies, and then incubated with horseradish peroxidase–conjugated anti-mouse IgG or anti-goat IgG secondary antibodies (1:8,500, Zymed), respectively. Signal was detected and developed using the ChemiLucent (Chemicon) system.
4
Western Blot Analysis of Sucrase Protein
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Protein samples were size-fractionated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, MA) by semi-dry blotting. The membranes were briefly rinsed with Tris-buffed saline containing 0.1% Tween 20 (TBS-T) and then incubated for 1 h at room temperature in TBS-T containing 5% skim milk (Difco, Detroit, MI). The blocked membrane was incubated with the primary antibody (Anti sucrase; Santa Cruz Biotechnology Inc., Santa Cruz, CA, 1:2,000) for 1 h at room temperature. Horseradish peroxidase-conjugated anti-mouse IgG (Zymed Laboratories Inc., South San Francisco, CA) was used as the secondary antibody, and proteins were visualized with ECL plus Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ) using LAS3000mini system (Fujifilm, Tokyo, Japan).
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