The cDNAs of RabA4d, RabA4b, RabD1, ROP6, and RabA1g were amplified from the Col-0, with the primers listed in the Supplemental Table . The primers used for site mutation of the constitutively active and dominant-negative forms of the Rab GTPases are also listed in the Supplemental Table . The PCR products were cloned into the pMD19-T vector and then cloned into the pGEX 4T-1 destination vector. The expression plasmids were transformed into Escherichia coli Rosetta cells for protein expression. Purification of GST-tagged recombinant proteins was performed as previously described by Xiang et al. (2007) (link). For lipid-protein overlay assays, PIP strips containing fifteen purified lipids (P-6001, Echelon Bioscience) were blocked in 3% (w/v) BSA in TBS-T (150 mM NaCl, 50 mM Tris, and 0.05% [v/v] Tween 20; pH 8.0) at RT for 3 h. The strips were then incubated with 2 μg/mL purified proteins in 10 mL of TBS-T with 3% (w/v) BSA at RT for 2 h. After incubation, the strips were washed three times for 10 min each with TBS-T, then incubated with 1:10,000 anti-GST primary antibody (Abmart, M20007) in 10 mL TBS-T at RT for 1 h, then washed three times for 10 min each with TBS-T and incubated with 1:10,000 secondary anti-mouse IgG (whole-molecule)-peroxidase antibody (Sigma Aldrich, A9044) in 10 mL of TBS-T at RT for 1 h. Images were obtained at RT using chemiluminescent signals.
Anti mouse igg whole molecule peroxidase antibody
Manufactured by Merck Group
Sourced in United States
The Anti-mouse IgG (whole-molecule)-peroxidase antibody is a laboratory reagent used for the detection and quantification of mouse immunoglobulin G (IgG) in various applications. It is composed of an anti-mouse IgG antibody conjugated to the enzyme peroxidase. The peroxidase label allows for the visualization and measurement of target mouse IgG molecules through colorimetric or chemiluminescent detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg whole molecule peroxidase antibody, by Merck Group (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Trans blot cell, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by Promega (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Luria bertani medium, by BD (1 mentions)
Hindiii, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by MedChemExpress (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Hnrnp k antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated beta actin monoclonal antibody, by Proteintech (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ibright imaging system, by Thermo Fisher Scientific (1 mentions)
19 protocols using anti mouse igg whole molecule peroxidase antibody
1
Characterization of Arabidopsis Rab GTPases
2
Immunoblotting of YFP-Fusion Proteins
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Apoplastic proteins were re-suspended in 1X Laemmli’s buffer (125mM Tris-HCl (pH 6.8); 1% SDS; 37.7% glycerol; 1% β-mercaptoethanol; 0.01% bromophenol blue), boiled for 5 minutes and used immediately for SDS-PAGE. Fifteen micrograms of total protein were loaded per well for 10% SDS-PAGE. After separation, the proteins were electrophoretically transferred to a Trans-Blot Transfer Medium membrane (Bio-Rad, 162–0112) using the Trans-Blot Cell (Bio-Rad) with a standard transfer buffer. Primary Anti-GFP monoclonal antibody (Covance, CA, USA, MMS-118P) at a dilution of 1:5000 and secondary Anti-Mouse IgG (whole molecule)-Peroxidase antibody (Sigma, A9044) at a dilution of 1:40000 were used for detection of YFP-fusion proteins. Membranes were treated with the HyGlo Quick Spray Reagent B for peroxidase activity (Denville, E2400) and immediately visualized by ChemiDoc™ XRS+ System (BioRad, 170–8265).
3
Western Blot Analysis of Renal Transporters
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Protein samples were extracted from the renal cortex tissues and quantified using a BCA protein concentration assay kit. Protein samples were separated on a 10% SDS-PAGE separation gel and transferred onto a 0.45 μm polyvinylidene fluoride membrane. The membranes were blocked with 5% skim milk in TBST buffer (Tris–HCl, NaCl, Tween 20) for 30 min and subsequently incubated with primary and secondary antibodies. Primary antibodies were as follows: rabbit monoclonal antibodies against XO (1:5000; ab109235), rabbit polyclonal antibodies against PNP (1:25,000; ab109559), URAT1 (1:1000; ab7816), GLUT9 (1:1000; ab223470) and OAT4 (1:1000; A7816); and mouse monoclonal antibodies against ABCG2 (1:1000; ab207732) and β-actin (1:5000; ab6276). Reactivity was detected using an anti-rabbit IgG (whole molecule)-peroxidase antibody (1:5000; Sigma A6154, St. Louis, MO) or anti-mouse IgG (whole molecule)-peroxidase antibody (1:5000; Sigma A4416, St. Louis, MO). Immunoreactive bands were detected with ECL reagents, and the densitometry analysis of the immunoblot results was conducted using ImageJ software (National Institutes of Health, Bethesda, MD).
4
Immunoblotting for Protein Purity
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Ten microliters (100 ng) of purified protein was used to analyze protein purity by immunoblotting using 1:10,000 anti- glutathione S-transferase (GST) primary antibody (Abmart, M20007) and incubated at RT for 1 h. Then, 1:10,000 secondary anti-mouse IgG (whole-molecule)-peroxidase antibody (Sigma Aldrich, A9044) was applied at RT for 1 h. Images were obtained using chemiluminescence detection.
5
Cloning and Expression of Recombinant Protein in E. coli
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The E. coli strains used were DH5α [F−Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17
phoA supE44 thi-1 gyrA96 relA1 λ−] and BL21 (DE3) [F−ompT hsdSB
gal dcm (DE3)] from Invitrogen (CA, USA). The plasmid used was pET28a(+) containing a 6-histidine tag (His-tag) at both the N- and C-terminal from Novagen (Darmstadt, Germany). T4 ligase and T4 buffer DNA ligase (2×) were purchased from Promega Corporation (WI, USA). The enzymes used (BamHI and HindIII) and the induction agent isopropyl β-d -1-thiogalactopyranoside (IPTG) were obtained from Thermo Scientific (MA, USA). The monoclonal anti-polyHistidine antibody produced in mouse, anti-mouse IgG (whole molecule) peroxidase antibody and 3′3′-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (MO, USA). Luria–Bertani (LB) medium was from BD (NJ, USA), and kanamycin from Gibco (MA, USA).
phoA supE44 thi-1 gyrA96 relA1 λ−] and BL21 (DE3) [F−ompT hsdSB
gal dcm (DE3)] from Invitrogen (CA, USA). The plasmid used was pET28a(+) containing a 6-histidine tag (His-tag) at both the N- and C-terminal from Novagen (Darmstadt, Germany). T4 ligase and T4 buffer DNA ligase (2×) were purchased from Promega Corporation (WI, USA). The enzymes used (BamHI and HindIII) and the induction agent isopropyl β-
6
Immunoblotting with Antibody Detection
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The monoclonal anti-FLAG M2, the polyclonal anti-HA, the anti-Mouse IgG (whole molecule) peroxidase antibody and the anti-Rabbit IgG (whole molecule) peroxidase antibody were from Sigma (USA). The polyclonal anti-RBM42 was from Abcam (UK). The monoclonal hnRNP K antibody was from Santa Cruz (USA). The HRP-conjugated Beta Actin monoclonal antibody was from Proteintech (USA).
The cells were washed with ice-cold PBS and then harvested in lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris, 1% Triton X-100) supplemented with protease inhibitor cocktail (MedChemExpress, USA). The lysates were incubated for 30 min in lysis buffer at 4°C and then centrifuged for 15 min at 16,000 ×g. The protein concentration was measured using BCA Protein Assay Kit (TaKaRa, Japan). The cell lysates were denatured using 5× SDS sample buffer and analyzed by immunoblotting with specific antibodies. Images were analyzed by the ImageJ software (1.8.0).
The cells were washed with ice-cold PBS and then harvested in lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris, 1% Triton X-100) supplemented with protease inhibitor cocktail (MedChemExpress, USA). The lysates were incubated for 30 min in lysis buffer at 4°C and then centrifuged for 15 min at 16,000 ×g. The protein concentration was measured using BCA Protein Assay Kit (TaKaRa, Japan). The cell lysates were denatured using 5× SDS sample buffer and analyzed by immunoblotting with specific antibodies. Images were analyzed by the ImageJ software (1.8.0).
7
Western Blotting of Recombinant Proteins
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Whole‐cell extracts were dissolved in NuPAGE LDS sample buffer (Thermo Fisher Scientific). 20, 10, 1, 0.5 and 0.1 µL of the extracts were loaded and fractionated on NuPAGE 12% Bis‐Tris gels (Thermo Fisher Scientific) under reducing conditions. Following SDS/PAGE, cell extracts were transferred onto poly(vinylidene difluoride) membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% (w/v) nonfat dry milk in the TBST buffer (50 mm Tris, 150 NaCl, 1% Tween 20, pH 7.6) for 1 h at room temperature. Following blocking, the membranes were incubated with GD‐26 IgG (1 mg·mL−1; 1 : 2000) or anti‐histidine tag antibody (clone HIS.H8; Merck Millipore; 1 : 2000) for 1 h at room temperature. After washing three times with the TBST buffer for 5 min, the membranes were incubated with a 1 : 100 000 dilution of anti‐mouse IgG (whole molecule)‐peroxidase antibody (Sigma‐Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The membranes were washed three times with the TBST buffer, developed with the chemiluminescent horseradish peroxidase substrate (Merck Millipore) and imaged by iBright Imaging Systems (Thermo Fisher Scientific). The same protocol was applied to western blotting on purified eGFP (5–20 ng).
8
Immunoblot Analysis of GST Proteins
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Immunoblot analysis was performed as described previously (Zhou et al., 2020 (link)). Briefly, immunoblot analysis was performed on polyvinylidene fluoride (PVDF) membranes (Hybond-P, GE) using an anti-glutathione S-transferase (GST) primary antibody (Abmart, M20007) and secondary anti-mouse IgG (whole-molecule)-peroxidase antibody (Sigma Aldrich, A9044). The blots were incubated at RT for 1 h. The signals were visualized using Amersham ECL Prime Western Blotting Detection Reagents (GE) and detected with Amersham Imager 600 (GE).
9
Western Blot Analysis Protocol
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Western blot analysis was performed as described previously (Miyashita et al., 2012 ). Briefly, cells were lysed with RIPA buffer (Nacalai Tesque, Shiga, Japan), and the cell lysates were then subjected to the polyacrylamide gel electrophoresis. Next, the separated proteins were transferred to the membranes, which were blocked for 1 h at room temperature with Tris–HCl‐buffered saline (TaKaRa Bio, Shiga, Japan) containing 0.05% Tween‐20 (T‐TBS) and 2.5% skim milk. After the transfer, the membranes were incubated for 1 h at room temperature with HRP‐conjugated anti‐human VASH1 mAb (4E12, 1:1000 dilution; Watanabe et al., 2004 ), anti‐human β‐actin monoclonal antibody (1:10000; Sigma‐Aldrich), or anti‐human α‐tubulin monoclonal antibody (1:1000 dilution; Merck Millipore, Billerica, MA, USA). After the membranes had been washed 3 times with T‐TBS, they were incubated for 1 h at room temperature with anti‐rat IgG (whole molecule)‐peroxidase antibody or anti‐mouse IgG (whole molecule)‐peroxidase antibody (Sigma‐Aldrich) as a secondary antibody. They were then washed again 3 times with T‐TBS, after which the blots were detected by an enhanced chemiluminescence method using an Immobilon Western HRP Substrate (Merck Millipore). The results were visualized by using an LAS‐4000 (Fuji Film, Tokyo, Japan).
10
Validating miR-221 Sponge Efficacy
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7.5×104 HepG2 cells were infected by rAd-199T-miR-221 sponge or corresponding control virus (MOI:10) and rAAV-miR-221 sponge or rAAV-control (MOI:100) in 24 well plate. Cells harvested at 72 h post infection and lysed by using RIPA lysis buffer (150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) (Sigma, St Louis, MO) with complete protease inhibitor cocktails (Sigma, St Louis, MO). Homogenates were then centrifuged at 13000 rpm for 15 min at 4°C and supernatants were collected and analyzed by western blotting. Protein concentration was measured using Bradford reagent and the BSA protein. 30 micrograms of total extracted protein were loaded on a 4-15% Mini-PROTEAN® TGX™ precast gels (Bio-Rad Laboratories, Hercules, CA94547). Blotting was performed for CDKN1B/ p27 (BD Transduction Laboratories™, 610242, dilution 1:1500). Beta-actin (Sigma Aldrich, A4700, dilution 1:1000) was used for normalization purposes. For recognition of the primary antibodies, anti-mouse IgG (whole molecule)–peroxidase antibody produced in rabbit (Sigma Aldrich, A9044 dilution 1:10000) were used. Protein bands were visualized using the ChemiDocTMMP Imaging System and Quatified using image lab 4.0 software (Bio-Rad Laboratories,Hercules,CA94547).
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