Cells were cultured on glass coverslips, washed 3 times with PBS and fixed in 4% (w/v) formaldehyde for 10 min at room temperature. Then the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 10% FBS in PBS for 2 h. the cells were incubated with 3G9 mAb and anti-His-tag (1:50, Abcam) primary antibody overnight at 4°C, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibody and rhodamine-conjugated goat-anti-mouse secondary antibody for 1h at room temperature. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Polysciences, Warrington, PA, USA) at 0.5mg/ml. Coverlslips were examined under a fluorescence microscope (Leica, Wetzlar, Germany).
Anti his tag
Manufactured by Abcam
Sourced in United States
Anti-His-tag is a lab equipment product used to detect and purify proteins with a histidine (His) tag. It provides a simple and reliable method for the identification and isolation of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Leica (1 mentions)
Cellquest software, by BD (1 mentions)
Rituximab, by Abcam (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Abcam (1 mentions)
4 chloro 1 naphthol, by Merck Group (1 mentions)
Hrp conjugated rabbit anti mouse igg antibody, by Abcam (1 mentions)
6 protocols using anti his tag
1
Immunofluorescence Staining of Cultured Cells
2
Quantifying scFv Binding to CD20 on Raji Cells
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In this experiment, the ability of selected scFv to bind to the native CD20 on Raji cells was measured. The Rituximab, a commercial anti-CD20 monoclonal antibody, was used as a positive control. First, cells were detached using 0.25% Trypsin–EDTA and counted using Hemocytometer. Then, for each antibody staining, 1 × 106 cells were washed twice with 1% PBS-BSA. All the antibody staining were performed for 1 h at RT in darkness. Cells were washed twice after each antibody staining. Next, cells were stained with 100 nM either Rituximab (Kindly provided by Tehran University Hospital) or scFv. Afterward, the secondary antibody was added to each tube, anti-Human (Abcam, USA) and anti-his tag (Abcam, USA) FITC conjugated for Rituximab and scFv stained samples, respectively. Next, one sample of each cell line was stained with Isotype as a negative control. Finally, the cells were analyzed by FACS-Calibur flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA, USA).
3
Co-Immunoprecipitation of FSIP1 and MRP1
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MDA-MB-431 cells that were stably infected with FSIP1 (His-tagged) lentiviral vector or empty vector was subjected to radio-immunoprecipitation assay (RIPA) buffer supplemented with proteinase inhibitor (Novagen, Darmstadt, Germany) in order to extract its proteins. The resultant cell lysate (containing 100 μg protein) was incubated with anti-His-tag (Abcam, Cambridge, Massachusetts, USA) or IgG (as a negative control) (Santa Cruz, Dallas, Texas, USA). Whole cell lysate (150 μg protein) and the immuno-precipitant of anti-His-tag, anti-MRP1 antibody or IgG were immunoblotted with either anti-FSIP1 or anti-MRP1 antibody to confirm the interaction of FSIP1 and MRP1. The lysate (6% input, 10 μg protein) was also used as a control. To validate the Co-IP results, immune complexes were then centrifuged to encourage precipitation before undergoing SDS-PAGE gel separation. Gel wells positive for candidate protein targets were extracted, subjected to in-gel digestion and subsequently underwent matrix-assisted laser desorption/ionization time-of-flight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry8 (link).
4
Protein Extraction and Western Blot Analysis
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The total protein in HCI-293 T cellswas extracted by RIPA lysis buffer containing protease inhibitor. The protein concentration was determined by the BCA method (Thermo Fisher Scientific, USA) following the manufacturer’s instructions.
Proteins were separated on a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was blocked in 5% skim milk for 2 h and then incubated with the primary antibody at 4℃overnight. After washing the PVDF membrane, it was incubated with the corresponding secondary antibody for 2 h at room temperature. Proteins were visualized on a gel imager using a SuperSignalTM West Pico PLUS chemiluminescentsubstrate (Thermo Fisher Scientific,USA). The primary antibodies used were as follows: anti-His tag (1:1000, Abcam)and anti-β-actin (1:2000, Abcam).
Proteins were separated on a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was blocked in 5% skim milk for 2 h and then incubated with the primary antibody at 4℃overnight. After washing the PVDF membrane, it was incubated with the corresponding secondary antibody for 2 h at room temperature. Proteins were visualized on a gel imager using a SuperSignalTM West Pico PLUS chemiluminescentsubstrate (Thermo Fisher Scientific,USA). The primary antibodies used were as follows: anti-His tag (1:1000, Abcam)and anti-β-actin (1:2000, Abcam).
5
Identifying MDGA2 interacting proteins
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The total protein of AGS and BGC823 cells stably transfected with MDGA2 (His-tagged) expression vector or empty vector was extracted in radioimmunoprecipitation assay (RIPA) buffer supplemented with proteinase inhibitor (Novagen, Darmstadt, Germany). Lysate (100 μg protein) was incubated with anti-His-tag (Abcam, Cambridge, Massachusetts, USA) or IgG (as a negative control) (Santa Cruz, Dallas, Texas, USA) (see online supplementary table S1). The immune complexes were precipitated by centrifugation and separated by SDS-PAGE. Candidate targets were excised from gels and subjected to protein identification by undertaking the in-gel digestion approach and using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry.8 (link)
Whole cell lysate (150 μg protein) and co-immunoprecipitation (Co-IP) precipitant by anti-His-tag, anti-DNA methyltransferase 1 associated protein 1 (DMAP1) antibody or IgG were immunoblotted with either anti-DMAP1 or anti-MDGA2 antibody to confirm the interaction of MDGA2 and DMAP1 that was identified by mass spectrometry. The lysate (6% input, 10 μg protein) was also used as a control.
Whole cell lysate (150 μg protein) and co-immunoprecipitation (Co-IP) precipitant by anti-His-tag, anti-DNA methyltransferase 1 associated protein 1 (DMAP1) antibody or IgG were immunoblotted with either anti-DMAP1 or anti-MDGA2 antibody to confirm the interaction of MDGA2 and DMAP1 that was identified by mass spectrometry. The lysate (6% input, 10 μg protein) was also used as a control.
6
Western Blot and Dot Blot Analysis of Recombinant Proteins
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For Western blot analysis the purified proteins were applied on a 10 % SDS-PAGE and transferred onto nitrocellulose membrane by using a semidry (Biorad, California, USA) system (Tris 12.5 mM, Glycine 96 mM, SDS 0.03 %, and methanol 10 % with pH 8.3). The membrane was incubated in the blocking buffer of 5 % skimmed milk at 4 ºC overnight. It was then incubated in a 1:1000 diluted primary antibody, anti-HisTag (Abcam, UK) or pan-dengue specific monoclonal antibody (cat no. MAB4043, Abnova), with gentle shaking for 2 hours at room temperature (RT). The membrane was washed three times with PBST (PBS containing 0.1 % Tween 20) and then incubated in secondary antibody, a 1:2000 dilution of HRP-conjugated rabbit anti mouse IgG antibody (Abcam, UK), with gentle shaking for 1 h at RT. After a 15 min washing with PBST, the detection was performed using diaminobenzidine (DAB) as a chromogenic substrate. Dot blotting analysis was performed using a very similar method like Western blotting. Briefly, recombinant proteins were dotted on nitrocellulose membrane strips and were reacted with diluted serum samples from the immunized animals. Blocking, antibody incubation, and detection methods were identical with Western blotting, except the serum dilutions were 1:10 and the chromogenic substrate was 4-Chloro-1-naphthol (Sigma-Aldrich, St. Louis, MO, USA).
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