Three millions of tumor cells (M14-wt, M14-A2, C8161 and SK-MEL-5) were lysate with 81µl of 2X Laemmil Sample Buffer (Bio Rad) and 9µl of 2-Mercaptoethanol (Fisher Scientific). Ten µl of total protein lysates were separated by Mini-PROTEAN TGX Stain-Free Gels (BIO RAD) electrophoresis. After protein transfer, membranes were incubated with 3µl of anti-tyrosinase (ThermoFisher, clone T311) or 15 µl of anti-MART-1 (Invitrogen, clone M2–7C10). After washes, membranes were incubated with 3µl of HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen). Staining for β-actin (Santa Cruz Biotechnolgy) was used as loading control. Membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
Hrp conjugated goat anti mouse igg h l secondary antibody
Manufactured by Thermo Fisher Scientific
The HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody is a laboratory reagent used to detect the presence of mouse immunoglobulin G (IgG) proteins in biological samples. It is conjugated with the enzyme horseradish peroxidase (HRP), which allows for colorimetric or chemiluminescent detection of the target IgG.
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Lab products found in correlation
Hrp conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse igg h l cross adsorbed secondary antibodies, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Anti tyrosinase, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
X ray film, by Fujifilm (1 mentions)
Srx 101a, by Konica Minolta (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Immobilion p, by Merck Group (1 mentions)
Ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Nupage reducing agent, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage antioxidant, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
9 protocols using hrp conjugated goat anti mouse igg h l secondary antibody
1
Protein Expression Analysis in Melanoma Cells
2
Western Blot Analysis of ASNS, RARS, and β-Actin
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Protein lysates were extracted with ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Thirty μg of protein lysates were separated using SDS–polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Immobilion-P, Millipore, IPVH00010). After blocking the membranes in 5% BSA in TBST (1× TBS (Cell Signalling); 0.1% Tween20 (Sigma)), the membranes were incubated overnight at 4 °C with primary antibodies either rabbit anti-ASNS antibody (Proteintech, 14681–1-AP) diluted 1:1,000 in 5% BSA (Sigma), mouse anti-β-Actin antibody (Millipore Sigma A5441) diluted 1:5,000 in 5% BSA (Sigma), and rabbit anti-RARS (Proteintech, 27344–1-AP) diluted 1:1,000 in 5% BSA (Sigma).
Primary antibodies were incubated in 5% BSA in TBST overnight at 4 °C. After washing the blots 3 times for 15 min each in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H+L) or HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen). Finally, the membranes were incubated with ECL western blot substrate (Thermo Scientific) for 1 min. X-Ray films (Fujifilm) were exposed to the western blot membranes and developed with a film processor (SRX-101A, Konica Minolta) and exposure.
Primary antibodies were incubated in 5% BSA in TBST overnight at 4 °C. After washing the blots 3 times for 15 min each in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H+L) or HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen). Finally, the membranes were incubated with ECL western blot substrate (Thermo Scientific) for 1 min. X-Ray films (Fujifilm) were exposed to the western blot membranes and developed with a film processor (SRX-101A, Konica Minolta) and exposure.
3
Quantifying Serum Antibody Titers
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The mice were bled by submandibular lancet 7 days after each immunization, and the serum was separated and pooled within each group. A 96-well Immulon 4HBX plate was coated with 2 µg/mL of MUC1 antigen in PBS and incubated overnight at 4 °C. The next day, the plate was washed five times with washing buffer (PBS with 0.1% Tween-20) and blocking buffer was added at a concentration of 15 µg/mL of BSA in PBS for 2 h. After washing five more times, different dilutions of mice sera were added and incubated for an hour at room temperature. After washing again, HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:10,000, Invitrogen) was added and incubated at room temperature. Finally, the plate was washed again before adding TMB substrate (BioFX, Inc., Eden Prairie, MN, USA) and the absorbance at 620 nm was measured.
4
Western Blot Analysis of E2 Protein
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Cells supernatants were diluted in NuPAGE LDS sample buffer and NuPAGE reducing agent (Invitrogen), heated at 70°C for 10 minutes, and loaded in a NuPAGE 4–12% Bis-Tris Gels (Invitrogen). Gel electrophoresis was carried out at 200V for 50 minutes in MOPS SDS running buffer containing NuPAGE antioxidant (Invitrogen). After gel electrophoresis proteins were transferred to a PVDF membrane by western blotting at 30V for 1 hour. After transfer, the membrane was rinsed with deionized water and incubated in blocking buffer (PBS 1% bovine serum albumin and 0.2% milk) for 30 minutes. The proteins were probed using E2-specific AP33 antibody [31 (link)] and HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen) and visualized by chemiluminescence.
5
Immunoblotting and Immunofluorescence Techniques
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Anti-Myc (2276S), anti-β-actin (8457S), anti-GluR1 (13185S), anti-NR1 (5704S), anti-PSD-95 (3450S), and anti-Map2 (4542S) antibodies are from Cell Signaling Technology. Anti-GFAP (16825-1-AP), anti-GluR2 (11994-1-AP), anti-NR2A (19953-1-AP), and anti-NR2B (19954-1-AP) antibodies are from Proteintech. Anti-Iba1 (019-19741), anti-GFP (M20004L), anti-NeuN (ab177487), anti-GluR3 (MAB5416), and anti-synaptophysin (S5768) antibodies are from Wako, Abmart, Abcam, Millipore, and Sigma, respectively. HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (31430), HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (31460), Alexa fluor 488-conjugated donkey anti-mouse IgG (H + L) secondary antibody (A-21202), Alexa fluor 488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (A-11008), and Alexa fluor 594-conjugated goat anti-rabbit IgG (H + L) secondary antibody (A-11012) are from Thermo Fisher Scientific.
6
Antibody Characterization for Diverse Targets
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The following antibodies were used in this study: Polyclonal rabbit anti-GFP (TP401; Torrey Pines; 1:2,000 for western blotting and 1:200 for immunostaining), anti-Rab10 (D36C4) Rabbit mAb (#8127, Cell signaling, 1:1000 for western blotting), anti-Rab11 antibody (ab3612, abcam, 1:1000 for western blotting), Recombinant anti-SNX5 antibody (ab180520, abcam, 1:1000 for western blotting). Purified Mouse anti-SNX1 (611482, BD biosciences, 1:100 for IF), anti-SNX6 mouse monoclonal antibody (D-5) (sc-365965, Santa Cruz, 1:1000 for western blotting), For pulldowns, Trap beads (nanobodies) were used. GFP-Trap_A (chromotek, gta-20) was used for GFP pulldowns. HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (ThermoFisher Scientific; 31430; 1:10,000) and polyclonal HRP-conjugated goat anti-rabbit IgG (ThermoFisher Scientific; 31460; 1:10,000) were used (incubated for 1 h at room temperature) to detect bound antibodies with Blotting detection kit WesternBrightTM ECL (advansta; K-12045-D50). Alexa Fluor 488–goat anti-rabbit IgG (H + L) (Invitrogen; A-11034) and Alexa Fluor 594–goat anti-mouse IgG (H + L) cross-adsorbed secondary antibodies (Invitrogen; R37121) were used for immunofluorescence. Janelia Fluor® 646 HaloTag® Ligand (Promega; Catalog number GA1120) was used for cargo transfer experiments.
7
Versatile Protein Modification Analysis
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Western blotting analysis was performed using standard procedures for whole-cell extracts. Antibodies used were antiacetyllysine mouse mAb (PTM-101) for Kac, anticrotonyllysine mouse mAb (PTM-502) for Kcr, antiphosphotyrosine mouse mAb (PTM-701) for Yph, anti-2-hydroxyisobutyryllysine rabbit pAb (PTM-801) for Khib, antiubiquitin rabbit pAb (PTM-1106) for Kub, and HRP-conjugated goat anti-Mouse IgG (H + L) secondary antibody (1:10,000, Thermo Fisher Scientific).
8
Immunofluorescence Staining Reagents
9
Antibody Validation for Protein Interactions
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The antibodies used in this study: polyclonal rabbit anti-VIPAS39 (20771-1-AP; proteintech; 1:2000), polyclonal rabbit anti-Rab11-FIP5 (NBP1-81855; Novus Biologicals;1:2,000), polyclonal rabbit anti-EHD1 (NBP2-56035; Novus Biologicals; 1:2,000), polyclonal rabbit anti-rabenosyn-5 (NB300-813; Novus Biologicals; 1:2,000), monoclonal mouse anti-myc (9E10) (1:3,000 for WB and 1:200 for Immunostaining; Sigma-Aldrich; M4439), polyclonal rabbit anti-GFP (TP401; Torrey Pines; 1:3,000 for WB and 1:200 for Immunostaining). For pulldowns Trap beads (Nano bodies) were used. GFP-Trap_A (chromotek, gta-20) for GFP pulldowns and myc-Trap_A (chromotek; yta-20) for myc pulldowns. HRP-conjugated goat anti-Mouse IgG (H+L) Secondary Antibody (Thermo Fisher scientific; 31430; 1:10,000), polyclonal HRP conjugated goat-anti-rabbit IgG (Thermo Fisher scientific; 31460; 1:10,000) were used (incubated for 1 h at RT) to detect bound antibodies with and an ECL system (ECL prime, Amersham, RPN2232). Alexa Fluor 488-goat anti-rabbit IgG (H+L) (Invitrogen; A-11034) and Alexa Fluor 594-goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibodies (Invitrogen; R37121) were used for immunofluorescence.
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