Cells (70% confluence) were plated on chamber slides and fixed with 4% paraformaldehyde at room temperature for 5 min. After fixation, cells were permeabilized with 0.1% Triton X-100 for 5 min. Then, cells were blocked with 10% FBS for 20 min at room temperature and incubated with the following primary antibodies at 4°C overnight: Anti-Flag tag (cat. no. F3165; Sigma-Aldrich; Merck KGaA; 1:1,000) and anti-HA tag (cat. no. 3724; Cell Signaling Technology, Inc.; 1:500). The anti-Mouse IgG (H+L) Alexa Fluor® 488 conjugate (cat. no. A-11001; Invitrogen; Thermo Fisher Scientific, Inc.; 1:200) or anti-Rabbit IgG (H+L) Alexa Fluor® 594 conjugate secondary antibodies (cat. no. A-11012; Invitrogen; Thermo Fisher Scientific, Inc.; 1:200) were added and incubated in the dark for 60 min at room temperature. Nuclear staining was performed with 50 ng/ml DAPI (cat. no. D21490; Invitrogen; Thermo Fisher Scientific, Inc.) in the dark for 5 min at room temperature. The fluorescence signal was imaged using a Zeiss LSM710 confocal microscope at ×400 magnification (Zeiss AG).
Anti ha tag
Manufactured by Merck Group
Sourced in United States
Anti-HA-tag is a laboratory reagent used to detect and purify proteins that have been engineered to contain a HA-tag. The HA-tag is a short amino acid sequence that can be added to a protein of interest, acting as a molecular label. Anti-HA-tag binds specifically to the HA-tag, allowing researchers to identify and isolate the tagged protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag tag, by Merck Group (4 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (2 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Immu mount, by Thermo Fisher Scientific (1 mentions)
Cell observer z1, by Zeiss (1 mentions)
Enhanced chemiluminescence western blotting detection kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Boster Bio (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti sox2, by Abcam (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti aff4, by Abcam (1 mentions)
Mouse anti myc tag, by Merck Group (1 mentions)
Fitc conjugated goat anti mouse igg secondary antibody, by Merck Group (1 mentions)
35 mm petri dishes, by MatTek (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti claudin 1, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti ha tag
1
Immunofluorescent Localization of Epitope-Tagged Proteins
2
Western Blot for Synchronized Malaria Parasites
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Trophozoite-stage cultures were enriched by the Percoll-sorbitol method to yield 96–99% infected cells and permit equivalent loading of synchronous parasites. Cells were lysed in chilled hypotonic lysis buffer (7.5 mM NaHPO4, 1 mM EDTA, pH = 7.5) with 2 mM PMSF prior to separation by SDS-PAGE (4 to 15% Mini-Protean TGX gel, Bio-Rad) and transfer to nitrocellulose membrane for immunoblotting. After blocking, primary antibodies were applied overnight in blocking buffer (anti-CLAG3, 1:2000 dilution; anti-HA tag, 1:1000 dilution, EMD Millipore). After washing to remove unbound antibody, horseradish peroxidate (HRP)-conjugated secondary antibody was applied (anti-mouse IgG, 1:10,000 dilution, Sigma-Aldrich) with Clarity Western ECL substrate (Bio-Rad). Binding was detected on Hyblot X-ray film. Band intensities were estimated from three independent trials using ImageJ software (https://imagej.nih.gov/ ) and normalization for hemoglobin loading with Ponceau S staining.
3
Immunofluorescence Staining of HA-tagged Proteins
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Cells were seeded on glass coverslips and fixed in a 1:1 methanol–acetone mixture for 2 min at room temperature, followed by blocking with 5% goat serum in PBS for 30 min. Cells were then washed in PBS and placed in a humid chamber to be stained with the primary antibody anti-HA tag at a concentration of 8 µg/mL (anti-rabbit, #H6908, Sigma-Aldrich, Deisenhofen, Germany) in PBS, with 0.1% BSA, for 1 h. After washing with PBS, cells were incubated with a Cy3-conjugated goat anti-rabbit secondary antibody (#115165146, Jackson ImmunoResearch, West Grove, PA, USA) at a concentration of 1.5 µg/mL in PBS, with 0.1% BSA, for 30 min at room temperature. After washing with PBS and staining with DAPI (200 nM in PBS, 5 min, followed by a PBS wash), coverslips were briefly dipped into 96% ethanol, air-dried, and mounted on glass slides with ImmuMount (Thermo Fisher Scientific, Waltham, MA, USA). Mounted cells were imaged using an inverted microscope (CellObserver Z1, Carl Zeiss, Jena, Germany) with a 63× NeoFluar oil immersion objective.
4
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed on ice for 30 min in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40), supplemented with protease inhibitor cocktail (Pierce Biotechnology). Aliquots of the lysates were subjected to dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were then transferred onto nitrocellulose membranes (Bio-Rad). The membranes were subsequently incubated with primary antibodies followed by a horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China). Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Thermo). The following antibodies for western blot were used in this study: anti-AFF4 (Abcam, 1:1000), anti-SOX2 (Abcam, 1:2000), anti-HA-tag (Sigma–Aldrich, 1:2000), anti-α-tubulin (Sigma–Aldrich, 1:5000).
5
Visualizing Protein Interactions in Cells
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To detect the expression of myc-pBCL-G and HA-pJAB1, SUVEC cells were seeded in 35-mm petri dishes (MatTek, MA, USA) and were co-transfected with pCMV-myc-pBCL-G and pCMV-HA-pJAB1 vectors. About 24 hours after transfection, cells were fixed with 4 % paraformaldehyde for 30 minutes at room temperature and permeabilized with 0.2 % Triton X-100/PBS for 5 minutes. Then, cells were incubated with mouse anti-myc tag (Sigma Aldrich, Saint Louis, USA) or anti-HA tag (Sigma Aldrich, Saint Louis, USA) monoclonal antibody (1:1000 dilution) for 1 hour at room temperature, followed by incubation with FITC-conjugated goat anti-mouse IgG secondary antibody (Sigma Aldrich, Saint Louis, USA, 1:100 dilution) for 1 hour at room temperature. After being rinsed with PBS, cells were observed using an inverted fluorescence microscope.
6
Co-Immunoprecipitation and Mass Spectrometry Analysis
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Cells were lysed on ice by IP lysis buffer, sonicated, and then centrifugated to collect the supernatant as previously described [16 (link)–18 (link)]. The protein supernatant was incubated with 2 µg of indicated antibodies or IgG at 4 °C overnight. The immune complexes were incubated with Pierce Protein A/G magnetic beads (Thermo Fisher Scientific) and then washed with IP wash buffer. The immune complexes were denatured, separated by SDS-PAGE, and stained by silver staining. The differentially protein bands were analyzed by mass spectrometry (Weifei Biotechnology Co., Shenzhen, China), and proteins of interest were detected by western blot. The antibodies used for co-IP included anti-HA-tag (H6908, Sigma-Aldrich), anti-Flag-tag (F1804, Sigma-Aldrich), and anti-Vimentin (5741 S, Cell Signaling Technology).
7
Immunohistochemical Antibody Reagents
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The following commercial monoclonal or polyclonal antibodies were utilized at appropriate concentrations: Anti-human/mouse EpCAM C-terminus (ThermoFisher Scientific); anti-Ki67 (abcam, Cambridge, MA, USA); anti-lysozyme (Dako, Via Real Carpinteria, CA, USA); anti-claudin-1 (Invitrogen); anti-claudin-3 (Invitrogen); anti-claudin-7 (Invitrogen); anti-claudin-15 (Invitrogen); anti-HA tag (Sigma, St Louis, MO, USA); anti-mouse β-Actin (AC-15, Sigma); anti-mouse claudin-1 (2H10D10, ThermoFisher Scientific); anti-FLAG tag (M2, Sigma). Several rabbit monoclonal antibodies (anti-murine EpCAM (clone E73) and anti-TROP2 (clone E69)) were generated by Epitomics, Inc. (Burlingame, CA, USA). via a contract.
8
Immunofluorescent Analysis of Rac1 Activation
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Antibodies used in this study were anti-Myc-tag (#2278), anti-GAPDH (#2118), anti-RhoGDI (#2564), all from Cell Signaling, anti-Rac1 (#610650, BD Transduction Laboratories), anti-mCherry (#NBP2-25157, Novus Bio) and anti-HA-tag (#H3663, Sigma)
For immunofluorescent staining, myc-Rac1 was stained using anti-Myc-tag (#2278, Cell Signaling) as primary antibody in combination with the secondary antibody Alexa 488 donkey anti-rabbit (#A32790, Invitrogen). In all stainings, nucleus was stained with DAPI (#62248, Thermo Fisher scientific) and F-actin with Acti-stain 670 phalloidin (#PHDN1-A, Cytoskeleton).
The inhibitors PR619 (#S7130) and MLN7243 (#S8341) were used at 2.5 μM concentration. MG132 (#S2619) was used at 5 μM concentration. All were from Selleck Chemicals.
For immunofluorescent staining, myc-Rac1 was stained using anti-Myc-tag (#2278, Cell Signaling) as primary antibody in combination with the secondary antibody Alexa 488 donkey anti-rabbit (#A32790, Invitrogen). In all stainings, nucleus was stained with DAPI (#62248, Thermo Fisher scientific) and F-actin with Acti-stain 670 phalloidin (#PHDN1-A, Cytoskeleton).
The inhibitors PR619 (#S7130) and MLN7243 (#S8341) were used at 2.5 μM concentration. MG132 (#S2619) was used at 5 μM concentration. All were from Selleck Chemicals.
9
Western Blotting of HBV Proteins
10
Immunoprecipitation and Mass Spectrometry Analysis
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Cells were lysed, sonicated and centrifuged to collect supernatant as previously described [21 (link)–23 (link)]. Then, the protein supernatant was incubated with 2.0 μg of an anti-HA-tag antibody (Sigma) or IgG overnight at 4 °C. Subsequently, the mixture was incubated with Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific). After washing with IP washing buffer, the collected immune complexes were separated by SDS-PAGE and then stained with silver staining. Mass spectrometry (MS) was performed by Wininnovate Bio (Shenzhen, China). The proteins of interest in the coimmunoprecipitate were detected by western blot analysis. The antibodies used were as follows: anti-HA-tag (H6908, Sigma), anti-Flag-tag (F1804, Sigma), anti-DNAJA4 (ab185553; Abcam) and anti-MYH9 (11128-1-AP; Proteintech).
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