Melanoma cells were seeded at a density of 6 × 105 cells ml−1 in 12-well plates and lysed the next day with LDS buffer 1× (Life Technologies). Lysates were denatured at 95 °C for 5 min and sonicated. SDS–PAGE and western blotting were performed using standard procedures. Membranes were visualized and bands quantified using an Odyssey Fc (LI-COR). Loading controls were run on the same blot. Individual 680 and 800 channels were co-visualized in greyscale on the same image. Primary antibodies were: LAP1 (1:1,000; #21459-1-AP), Lamin A/C (1:1,000, #10298-1-AP), Lamin B1 (1:1,000, #12987-1-AP), Lamin B2 (1:1,000, #10895-1-AP) from Proteintech; pThr18/Ser19-MLC2 (1:750, #3674), MLC2 (1:750, #3672) from Cell Signaling Technology; GAPDH (1:10,000, #MAB374) from Merck. Secondary antibodies were: IRDye 680RD goat anti-rabbit IgG (1:10,000, #925-68071) and IRDye 800RD goat anti-mouse IgG (1:10,000, #925-32210) from LI-COR.
Irdye 800rd goat anti mouse igg
Manufactured by LI COR
The IRDye 800RD goat anti-mouse IgG is a secondary antibody reagent designed for detection and quantification applications. It consists of the IRDye 800RD fluorescent dye conjugated to a goat-derived polyclonal antibody that specifically binds to mouse immunoglobulin G (IgG). This product is intended for use in various immunoassay techniques, such as Western blotting, immunohistochemistry, and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680rd goat anti rabbit igg, by LI COR (3 mentions)
Lamin b1, by Proteintech (2 mentions)
Lamin b2, by Proteintech (2 mentions)
Pthr18 ser19 mlc2, by Cell Signaling Technology (2 mentions)
Odyssey fc, by LI COR (2 mentions)
Gapdh, by Merck Group (2 mentions)
Lamin a c, by Proteintech (1 mentions)
Odyssey v3, by LI COR (1 mentions)
H3k9me2, by Thermo Fisher Scientific (1 mentions)
Tgx fastcast gel, by Bio-Rad (1 mentions)
Cul4a, by Proteintech (1 mentions)
H3k9me1, by Thermo Fisher Scientific (1 mentions)
H3k27me3, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Lds buffer 1x, by Thermo Fisher Scientific (1 mentions)
Mini protean tgxtm gel, by Bio-Rad (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Odyssey clx, by LI COR (1 mentions)
4 protocols using irdye 800rd goat anti mouse igg
1
Western Blot Analysis of Melanoma Cells
2
Epigenetic Changes in Huntington's Disease
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Protein lysates of NPC cell pellets (Control, 45Q, and 81Q) were prepared using RIPA lysis buffer (RIPA Buffer, 1 mM PMSF, 5 μm Z-VAD, 1 mM NaVan, and 1 × Complete Protease Inhibitor Mixture tablets). Protein lysates were sonicated for 2 cycles of 30 s on, 30 s off. 35 μg of protein lysates were separated on 10% TGX FastCast gel (Biorad) and transferred on nitrocellulose membranes (Biorad). The following primary antibodies were used: CUL4A (1:1000; Proteintech Cat# 14851-1-AP, RRID:AB_2261175 ), H3 (1:2500, Abcam Cat# ab1791, RRID:AB_302613 ), H3K27me3 (1:500, Millipore Cat# 07-449, RRID:AB_310624 ), H3K9me1 (1:500; Thermo Fisher Scientific Cat# MA5-33385, RRID:AB_2815523 ), H3K9me2 (1:500; Thermo Fisher Scientific Cat# 720092, RRID:AB_2532802 ), and β-actin (1:5000; Sigma-Aldrich Cat# A5441, RRID:AB_476744 ) (Supplemental Table S1 ). Antibody validation proved by venders. Membranes were incubated with primary antibodies at 4 °C overnight. The following secondary antibodies (1:5000) were used: IRDye® 680RD Goat anti-Rabbit IgG and IRDye® 800RD Goat anti-Mouse IgG (LI-COR). The membrane was imaged using the LI-COR Imaging System and Odyssey V3.0 software (LI-COR), followed by intensity analysis with GelAnalyzer. Statistical significance was tested using a two tailed t-test with a P-value threshold of 0.05.
3
Quantification of Melanoma Cell Proteins
4
Stimulation of CXCR2 signaling in HEK293 cells
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HEK293-CXCR2 cells were plated in a 6 well-plate at a density of 40,000 cells per well in reduced serum media (0.5% FBS). Approximately 24 hours later, cells were serum-starved in DMEM supplemented with 1% Pen/Strep at 37 °C with 5% CO2 for 2 hours. Following serum starvation, cells were stimulated with indicating concentrations of CXC-ligands for 5 mins and stopped with ice-cold PBS. Subsequently, 200 mL of 1 x Laemmli buffer was added per well. Lysates were collected, resolved on 4 to 20% Mini-protean TGXTM Gel (4561094, Bio-Rad) and transferred onto a nitrocellulose membrane (1215458, GVS). After transfer, the membrane was incubated with TBS buffer supplemented with 0.1% Tween 20 (TBS-T) and 3% BSA at room temperature for 1 hour on a shaker. The membrane was probed with primary antibodies (anti-phosphoERK1/2 (Cell Signaling 4370S)), anti- anti-ERK1/2 (Cell Signaling 4696S) diluted 1:1000 in TBS buffer supplemented with 0.1% Tween 20 and 3% BSA at 4 °C overnight. The next day, the membrane was washed with TBS-T four times and probed with the secondary antibodies (goat anti rabbit IgG, 680 RD (926–68071, LiCor), IRDye® 800RD Goat anti-Mouse IgG (926–32210, LiCor) at 1:10,000 dilution at room temperature for 1 hour. After another four washes with TBS-T, protein bands on the membrane were visualized using Li-Cor Odyssey CLx and analyzed using StudioLite software [20 (link)].
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