At time of collection, HUVEC were washed three times for 5 min in PBS with Ca2+ and Mg2+ containing 0.2% (wt/vol) BSA. Cells were then incubated with 3 μM ALOD4 in basal EBM2 media containing 0.2% (wt/vol) BSA for 1 hr at 4 °C. The unbound proteins were removed by washing three times with PBS with Ca2+ and Mg2+ for 5 min each. Cells were then lysed and prepared for SDS-PAGE and immunoblotting. ALOD4 was probed on nitrocellulose gels using anti-6X His (abcam) antibody at 15 kDa. A similar method was used for OlyA binding.
Anti 6x his
Manufactured by Abcam
Anti-6X His is a laboratory product that can be used for the detection and purification of proteins that have been tagged with a 6-histidine (6xHis) sequence. It is a tool used in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd98, by Thermo Fisher Scientific (2 mentions)
Anti cd98, by Abcam (2 mentions)
Iblot2, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit, by Jackson ImmunoResearch (1 mentions)
Hoechst, by Merck Group (1 mentions)
Te2000, by Nikon (1 mentions)
6 protocols using anti 6x his
1
HUVEC Binding Assay for ALOD4 and OlyA
2
CD98 Immunoprecipitation of Env-Treated T Cells
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A total of 293 T cells were transfected with CD98 (OriGene, Rockville, MD; RC200178) using Lipofectamine 3000. Cells were incubated for 48 hours and treated with 100nM Env protein for 1 hour at 37°C. One million cells/immunoprecipitation were lysed using radioimmunoprecipitation assay buffer and immunoprecipitated overnight at 4°C using anti‐IgG (Cell Signaling Technology, Danvers, MA; 5412S), anti‐6XHis (Abcam, Cambridge, UK; 9108), or anti‐CD98 (Invitrogen, PA5‐81032). Complexes were precipitated using Protein A/G beads (Pierce, Rockford, IL) and washed 2× with TNE300 + 0.1% NP‐40, 2× with TNE50 + 0.1% NP‐40, and 2× with PBS + 0.05% Triton X‐100 (PBS‐T). Beads were resuspended in 30μl Tris‐Glycine Sample Buffer supplemented with 2‐mercaptoethanol, incubated at 95°C for 1 hour, and run on a 4 to 20% Tris‐Glycine gel (Invitrogen). The gel was transferred on to a polyvinylidene difluoride membrane using an iBlot2 (Thermo Fisher Scientific, IB24001), blocked for 1 hour with 5% milk in PBS‐T, then incubated overnight with anti‐CD98 (Abcam, 108300). The membrane was incubated with appropriate secondary antibody for 1 hour at room temperature and visualized.
3
CD98 Interaction Analysis in 293T Cells
4
ALOD4 and OlyA Binding Assay
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At time of collection, HUVEC were washed three times for 5 min in PBS with Ca2+ and Mg2+ containing 0.2% (wt/vol) BSA. Cells were then incubated with 3 μM ALOD4 in basal EBM2 media containing 0.2% (wt/vol) BSA for 1 h at 4°C. The unbound proteins were removed by washing three times with PBS with Ca2+ and Mg2+ for 5 min each. Cells were then lysed and prepared for SDS-PAGE and immunoblotting. ALOD4 was probed on nitrocellulose gels using anti-6X His (Abcam) antibody at 15 kDa. A similar method was used for OlyA binding.
5
Immunocytochemical detection of His-tagged proteins
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After appropriate treatments, culture medium was removed and cells were washed with PBS. Cells were fixed with 4% paraformaldehyde (pfa) for 20 min at room temperature. Cells were washed three times with PBS, before being blocked (5% Donkey Serum and 0.3% triton in PBS) at room temperature (RT) for 2 h. Primary antibody staining was carried out in blocking solution using Anti-6xHIS (1:500; Abcam; ab9108), at 4°C overnight. Secondary staining was carried out in blocking solution using Alexa Fluor 488 Anti-Rabbit (1:400; Jackson ImmunoResearch; 711-545-152) for 2 h at RT. Nuclei were stained with DAPI 1:50,000 in PBS for 10 min at RT.
6
Immunofluorescence Imaging of hERG1 in Cardiac Tissue
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Immunofluorescence (IF) on cells was performed following the protocol previously described in ref. 27 . For IF on cardiac tissue, fresh human tissue from healthy atrium was obtained by the Department of Cardiac Surgery of the Azienda Ospedaliero-Universitaria Senese, snap frozen in liquid nitrogen, and cut with a cryostat in 5-mm sections. After 2 hours of blocking in PBS with 10% BSA, sections were incubated for further 2 hours with either scDb-hERG1-b1 or scFv hERG1 antibodies (20 mg/mL and 200 mg/mL final concentrations), followed by 1 hour with anti-6xHis (Abcam) and then 1 hour with antimouse Alexa Fluor 488 (Thermo Fisher Scientific). Incubation with mAb hERG1 was performed overnight at a final concentration of 1 mg/mL. All incubations were performed at room temperature. Nuclei were stained with Hoechst (1:1,000 in PBS, 45 minutes; Merck Sigma). Images were captured using confocal microscope Nikon TE2000.
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