HepG2 and 293T cell lines were transfected with 10 μg of either pcDNA-HA-hPGC1α or pcDNA-HA-hPGC1β expression vectors. Twentyfour hours post transfection, cells were treated with 5 μM MG132 (Sigma-Aldrich) (Trausch-Azar et al., 2010 (link)). An additional 24 hours later, cells were lysed in 300 μl Laemmli (2x) buffer (65.8mM Tris-HCL, pH 6.8, 26.3% (w/v) glycerol, 2.1% SDS, 10.0% (v/v) 2-mercaptoehtanol). Total cellular protein (25 μg) was resolved by 4-8% SDS-PAGE and transferred to Immobilon PVDF membrane (Millipore) (Mao et al., 2013 (link)). The membranes were probed with HA-Tag rabbit monoclonal antibody (Cell Signaling Technology #3724, 1:1000 dilution) and GAPDH rabbit monoclonal antibody (Cell Signaling Technology #5174, 1:2000 dilution) followed by incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (Cell Signaling Technology #7074, 1:2000). HA-tagged and GAPDH polypeptides were detected using enhanced chemiluminescence (Thermo Fisher Scientific #34080) as described by the manufacturer and quantitated using the ChemiDoc MP Imaging System (BioRad).
Ha tag rabbit monoclonal antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The HA-Tag rabbit monoclonal antibody is a laboratory reagent used to detect the presence of the HA (Hemagglutinin) tag in recombinant proteins. The HA tag is a common epitope tag used to facilitate the identification and purification of proteins expressed in various systems.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (1 mentions)
Gapdh rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phospho mdm2 ser166, by Cell Signaling Technology (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Ab11251, by Abcam (1 mentions)
Rotenone, by Merck Group (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Ab1218, by Abcam (1 mentions)
96 well plate, by USA Scientific (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Spectramax m5, by PerkinElmer (1 mentions)
Streptavidin hrp, by PerkinElmer (1 mentions)
4 protocols using ha tag rabbit monoclonal antibody
1
PGC-1 Isoform Overexpression in Cells
2
Antibody Generation and Validation
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NgBR rabbit monoclonal antibody (CloneID: EPR8668) was generated by Epitomics (Burlingame, CA, USA) as a collaboration project. NgBR rabbit polyclonal antibody was generated as described previously [19 (link)] and was used for immunohistochemistry analysis. Rabbit polyclonal antibodies for phospho-AKT (Ser473), total Akt, Phospho-MDM2 (Ser166), cyclinD1, p21, phos-Rb (Ser807/811), HA-Tag rabbit monoclonal antibody, Histone H3, and β-actin and all the secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA). Rabbit polyclonal antibodies for p53, ubiquitin, CDK6, and β-tublin were purchased from ProteinTech (Chicago, IL, USA). Rabbit polyclonal antibody for total MDM2 was purchased from Anbo Biotechnology (San Francisco, CA, USA).
3
Profiling Protein Interactions and Apoptosis
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The antibodies against different tags and proteins used for immunoprecipitation and immunoblotting were as follows: GFP antibodies (rabbit polyclonal, ab290; mouse monoclonal ab1218, Abcam); DJ-1 antibodies (rabbit monoclonal, #5933, Cell Signaling; mouse monoclonal, ab11251, Abcam); mouse monoclonal Bag5 antibody (ab56738, Abcam); Myc antibodies (rabbit polyclonal, #2272, Cell Signaling; mouse monoclonal, #2276, Cell Signaling); HA-Tag rabbit monoclonal antibody (#3724, Cell Signaling); Hsp70 rabbit monoclonal antibody (#4876, Cell Signaling); mouse monoclonal ANTI-FLAG® M2 antibody (F1804, Sigma-Aldrich). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Thermo Scientific (V13242). Cycloheximide (R750107), rotenone (R8875), and rhodamine 123 (R8004) were purchased from Sigma.
4
Modified Tat ELISA for Protein Detection
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Since we couldn’t find a good commercial Tat ELISA kit. We used a modified Tat ELISA protocol. Briefly, 96-well plate (USA Scientific) was coated with 0.2 μg HA-tag rabbit monoclonal antibody (Cell Signaling) per well. Wash the wells with TBST and block the wells with blocking buffer (Licor) for 1 h at room temperature. Add 100 μL sample to specific wells and incubate at 4 °C overnight. After washing with TBST, Anti-HIV1 tat antibody (Biotin) (Abcam) in blocking buffer with 1:100 dilution was added to each well and incubate at room temperature for 2 h. Wash the wells with TBST for three times. Incubate with 100 μl diluted Streptavidin-HRP (1:100 dilution) (PerkinElmer) for 30 min at room temperature. After washing with TBST for 3 times, incubate with 100 μl OPD (O-Phenylenediamine Dihydrochloride) (PerkinElmer) for 30 min at room temperature, add 100 μl stop solution (PerkinElmer) to terminate the reaction, measure the absorbance at wavelength 490 nm using SpectraMax M5.
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