Protein concentration was measured by staining with amido black assay (Merck) according to the manufacturer's specifications. Per sample, 50 μg of proteins and 5 μl of PageRuler Prestained Protein Ladder (SM0671, Fermentas) were fractionated by electrophoresis through 12% SDS-polyacrylamide gels (SDS-PAGE) and transferred to Protran nitrocellulose membranes (Whatman) by semi-dry blotting using Tris-base, Glycerol, (Merck), 10 % SDS, methanol (J.T.Baker, The Netherlands) in double-distilled water for 30 min with a constant current at 100 mA per membrane. The membranes were blocked with 5% non-fat dry milk (AppliChem GmbH) in TBST (Tris-buffered saline (TBS) + 0.05% Tween®-20, Serva) or with 3% BSA in TBST (Sigma-Aldrich, München). For the detection of active RAL and RAS proteins, we used the following primary mouse monoclonal antibodies: RALA included in STA-408-CB and pan-RAS included in STA-400-CB pull-down assays, RALB: #3523, KRAS: #2146 and beta-tubulin: #TA801672 (Cell Signaling). Anti-mouse IgG-Horseradish Peroxidase (HRP)-conjugate (1:5000 dilution, 325-035-045, Dianova) or anti-rabbit IgG-HRP-conjugate (1:10, 000 dilution; 7074, Cell Signaling) were used as secondary antibody. Protein bands were visualized using the ECL chemoluminescence detection system (GE Health Care) on X-ray films (Amersham Hyperfilm e).
Anti rabbit igg hrp conjugate
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-rabbit IgG-HRP conjugate is a secondary antibody coupled with horseradish peroxidase (HRP) that specifically binds to rabbit primary antibodies. It is used to detect and visualize the presence of rabbit-derived proteins in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg hrp conjugate, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Criterion tris hcl gel, by Bio-Rad (3 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti haptoglobin, by Calibre Scientific (2 mentions)
Anti adam10, by Abcam (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Anti cd63, by BD (2 mentions)
Anti cd81, by BD (2 mentions)
Nr2f2 ab1, by Abcam (2 mentions)
Nr2f2 ab2, by Cell Signaling Technology (2 mentions)
Anti fog2, by GeneTex (2 mentions)
Donkey anti mouse igg hrp conjugate, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Hybond ecl membrane, by GE Healthcare (2 mentions)
Ecl plus, by GE Healthcare (2 mentions)
Pd 10 column, by GE Healthcare (2 mentions)
Tween 20, by Serva Electrophoresis (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti rabbit igg hrp conjugate
1
Protein Concentration Measurement and Western Blot Analysis
2
Western Blot Antibody Protocol
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Primary antibodies were used at 1–2 μg ml−1 for immunoblotting: anti-ADAM10 (mouse monoclonal, Abcam ab73402), anti-CD63 (mouse monoclonal, BD Biosciences 556,019), anti-CD81 (mouse monoclonal, BD Biosciences 555,675), anti-Haptoglobin (rabbit polyclonal, Biozol, GTX 112,962–25). The following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 goat anti-rabbit IgG (both from Life Technologies) and anti-mouse IgG-HRP conjugate and anti-rabbit IgG-HRP conjugate (both from Cell Signaling).
3
Immunoblotting Analysis of Nr2f2 in Hypertensive Rats
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Antibodies were from the following sources: Anti-Nr2f2 mouse monoclonal (Nr2f2-Ab1, Abcam, 1:2000 dilution), anti-CoupTF II (Nr2f2-Ab2, Cell signaling, 1:500 dilution), anti-Fog2 (GeneTex, 1:1000 dilution), donkey anti-mouse IgG-HRP conjugate (SC-2020, 1:5000 dilution), anti-rabbit IgG-HRP conjugate (Cell Signaling, 1: 5000 dilution). Samples were homogenized in ice-cold RIPA lysis buffer with protease inhibitor cocktail (Pierce). Immuno-blotting for Nr2f2 was carried out using tissue lysates from Nr2f2mutant and wild-type hypertensive rat homogenates. In brief, forty microgram of proteins were boiled with Laemmli sample buffer (Bio-Rad) for 5 minutes at 95° C. Protein samples were resolved using 4–15% Criterion Tris-HCl Gel (Bio-Rad) at room temperature, and transferred on to PVDF membrane (Millipore), blocked with 5% fat free milk powder in PBST and incubated with anti-Nr2f2 and secondary antibodies conjugated with horseradish peroxidase. Immunoreactivity was detected by autoradiography using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Full-length blots are shown in Supplementary Figure 1 .
4
Extraction and Detection of L1CAM
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Choi-CK cells were incubated with Ab417 (10 μg/mL) for the indicated time, and membrane protein fractions were obtained with MEM-PERTM Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). The obtained protein samples were quantified by a BCA protein quantification assay and subjected to SDS-PAGE. After transfer to nitrocellulose membrane and blocking with Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk (BD Biosciences), L1CAM was detected using a mouse mAb binding to human L1CAM [21 (link)] and goat anti-mouse IgG-HRP conjugate (1:8,000, v/v, Thermo Fisher Scientific). Pan-cadherin was used as a loading control with the Pan-cadherin rabbit mAb (1: 2,500, v/v, Cell Signaling Technology, USA) and anti-rabbit IgG-HRP conjugate (1:5,000, v/v, Cell Signaling Technology). Finally, immunoreactive bands were visualized using a SuperSignalTM West Femto Maximum Sensitive Substrate (Thermo Fisher Scientific).
5
Western Blot Analysis of Biotinylated Proteins
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Samples of cell lysates were analyzed by Western blotting for biotinylation. After SDS-PAGE under non-reducing conditions, proteins were transferred to a nitrocellulose membrane and stained with Ponceau S (Sigma Aldrich) to monitor protein loading. Membranes were blocked in 5% BSA (w/v) in TBST for 1 hour at room temperature. Blocked membranes were then incubated with anti-biotin D5A7 antibody (Cell signaling) diluted 1:2,000 in TBST containing 1% BSA (w/v) overnight at 4°C. The membrane was then washed once with TBST for 5 minutes at room temperature, and then incubated with anti-rabbit IgG, HRP conjugate (Cell Signaling) diluted 1:5,000 in TBST containing 1% BSA for 1 hour at room temperature with shaking. Prior to chemiluminescent detection, blots were washed with TBST three times for 10 minutes each time. Western blots were developed with Western Lightning® Plus-ECL, Enhanced Chemiluminescence Detection Kit (PerkinElmer).
6
Immunoblotting Analysis of Nr2f2 in Hypertensive Rats
7
Characterization of Rat Kidney Proteins
8
Detailed Western Blotting Protocol
9
Modulation of Hypoxia Signaling Pathways
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Dichloroacetate was purchased from Sigma-Aldrich (347795). Used at 4 mM final concentration. EUK134 was purchased from Sigma-Aldrich (SML0743) and used at 1 mM final concentration. NEMO-binding domain peptide (480025) from Millipore. Rotenone and antimycin were purchased from Sigma-Aldrich. Primary antibodies used were HIF-1α (1:500 in 5% non-fat milk and TBST for Western, 1:100 in 5% BSA and TBST for immunofluorescence, Cayman, Ann Arbor, MI, 10006421, RRID:AB_10099184 ), SLC2A1 (1:1000 in 5% BSA and TBST, ProteinTech, Rosemont, IL, 21829–1-AP, RRID:AB_10837075 ), HK2 (1:1000 in 5% BSA and TBST, Cell Signaling, Danvers, MA, C64G5, RRID:AB_2232946 ), β-actin (1:5000 in 5% BSA and TBST, Abcam, ab6276, RRID:AB_2223210 ), PDK1 (1:1000 in 5% non-fat milk and TBST, Cell Signaling, C47H1, RRID:AB_1904078 ), NOX4 (1:500 in 5% milk and TBST for Western, 1:50 in 5% BSA and TBST for immunofluorescence, Santa Cruz, Dallas, TX, sc-30141, RRID:AB_2151703 ), CD31 (BD, Franklin Lakes, NJ 550274, RRID:AB_393571 ), EPAS1 (1:1000 in 5% BSA and TBST, Novus Biologicals, Littleton, CO, NB100-122, RRID:AB_10002593 ). Secondary antibodies for Western blotting: Goat anti-mouse IgG, HRP conjugate (1:5000 in 5% non-fat milk and TBST, Calbiochem, 401253, RRID:AB_437779 ). Anti-rabbit IgG, HRP conjugate (1:3000 in 5% non-fat milk and TBST, Cell Signaling, 7074S, RRID:AB_2099233 ).
10
Purification of ASCT2 Transporter Protein
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The P. pastoris wild type strain (X-33), the pPICZB vector, zeocin, Ni-NTA agarose resin were from Invitrogen; anti-rabbit IgG HRP conjugate from Cell Signaling; PD-10 columns, ECL plus, Hybond ECL membranes were from GE Healthcare; L-[3H]Glutamine was from Perkin Elmer; anti-ASCT2 (rabbit) was from Millipore; conjugated anti-His antibody, C12E8, Cholesteryl hemisuccinate, Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcholine from egg yolk), Sephadex G-75, L-glutamine, methyl-β-cyclodextrin (MβCD) and all the other reagents were from Sigma-Aldrich.
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