Protein lysates were prepared in radioimmunoprecipitation assay buffer and quantified with the Bradford assay (Bio-Rad). Electrophoresis was performed in precast, NuPage 4 to 12% bis-tris protein gels before transferring into polyvinylidene difluoride membranes using the Trans-Blot Turbo transfer system (Bio-Rad). Before blocking and antibody incubation, membranes were stained with Ponceau S reagent (Sigma-Aldrich, #P3504) to visualize protein bands and cut at specific sizes so different antibodies could be tested at the same time. Western blots were performed using antibodies against H3K27me3 (0.4 μg/ml; Sigma-Aldrich, rabbit polyclonal), Isl1 (1:10,000; rabbit monoclonal, Abcam), Hsp90 (1:2000; rabbit polyclonal, Cell Signaling Technology), Gapdh (1:2000; rabbit monoclonal, Cell Signaling Technology), a goat anti-rabbit immunoglobulin G, and horseradish peroxidase–linked antibody (1:2000; Cell Signaling Technology). Densitometry analysis of Western blots was performed using Fiji ImageJ.
Goat anti rabbit immunoglobulin g
Manufactured by Cell Signaling Technology
Goat anti-rabbit immunoglobulin G (IgG) is a secondary antibody that binds to primary rabbit antibodies. It is used in various immunoassays and detection techniques to amplify and visualize the signal from rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bradford assay, by Bio-Rad (1 mentions)
H3k27me3, by Merck Group (1 mentions)
Ponceau s reagent, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti ha antibody, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Corning (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
3 protocols using goat anti rabbit immunoglobulin g
1
Western Blot Analysis of Protein Lysates
2
HEK293T Cell Culture and Transfection
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Human embryonic kidney cells (HEK293T, ATCC, Cat #CRL-3216) were cultured in Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, Invitrogen, Cat#11965092) supplemented with 10 % (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific, Invitrogen, Cat#10082147) and Antibiotic-Antimycotic solution (Thermo Fisher Scientific, Invitrogen, Cat# 15240062) at 5% CO2 and 37 °C. Cells were seeded on poly-D-lysine (Corning, Cat# 354210) coated 24-well trays for 16 h in the basal medium and transfected with 0.5–0.8 μg DNA/well by lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen, Cat# 11668019) in OPTI-MEM medium.
For western blot, all the samples were heated at 96 °C for 6 min before loading on SDS-PAGE gel. The protein bands were transferred to PVDF membranes (Millipore, Cat# PVH00010). After being blocked with 5% nonfat dry milk, the membranes were incubated with anti-HA antibody (Thermo Fisher Scientific, Cat#26183) at 4 °C overnight. Bound primary antibodies were detected with HRP-conjugated goat anti-mouse immunoglobulin-G at 1:5000 (Cell Signaling Technoloy, Cat# 7076S) or goat anti-rabbit immunoglobulin-G at 1:2500 (Cell Signaling Technology, Cat# 7074S) by chemiluminescence (VWR, Cat# RPN2232). The images of the blots were taken using a Bio-Rad ChemiDoc Imaging System.
For western blot, all the samples were heated at 96 °C for 6 min before loading on SDS-PAGE gel. The protein bands were transferred to PVDF membranes (Millipore, Cat# PVH00010). After being blocked with 5% nonfat dry milk, the membranes were incubated with anti-HA antibody (Thermo Fisher Scientific, Cat#26183) at 4 °C overnight. Bound primary antibodies were detected with HRP-conjugated goat anti-mouse immunoglobulin-G at 1:5000 (Cell Signaling Technoloy, Cat# 7076S) or goat anti-rabbit immunoglobulin-G at 1:2500 (Cell Signaling Technology, Cat# 7074S) by chemiluminescence (VWR, Cat# RPN2232). The images of the blots were taken using a Bio-Rad ChemiDoc Imaging System.
3
Western Blot Analysis of HPSE
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Whole cell lysates were prepared using RIPA buffer (Bio-Rad Laboratories, Inc.) containing the protease inhibitor PMSF (Invitrogen; Thermo Fisher Scientific, Inc.). The protein concentrations were determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). A total of 20 µg protein per lane was loaded and resolved by 10% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. Following washing, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature. Immunoreactive proteins were detected using enhanced chemiluminescence (Bio-Rad Laboratories, Inc.). The relative band intensity was quantified using ImageJ software (National Institutes of Health). The following antibodies were used: Anti-HPSE (1:500; cat. no. ab85543; Abcam), anti-GAPDH (1:2,000; cat. no. 2118; Cell Signaling Technology, Inc.) and goat anti-rabbit immunoglobulin G (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.).
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