To detect the expression of exogenous HA tagged PGRMC1 in Fig. 1 e, cells were seeded on coverslips on a six well plate. The cells were washed with ice-cold PBS, mildly fixed with 3.7% formaldehyde for 5 min at 4 °C. The cells were then permeabilized with ice-cold 100% methanol for 10 min at − 20 °C, followed by overnight incubation with anti-HA tag antibody (Sigma, H3663). The cells were washed extensively and incubated with FITC conjugated secondary antibody (Sigma, F8521) in dark for 1 h at 4 °C. Cells were washed three times with PBS and counterstained with DAPI mounting solution. Images were captured using a Nikon Ti Eclipse Confocal microscope (Nikon Australia Pty Ltd).
Anti ha tag antibody
Manufactured by Merck Group
The Anti-HA tag antibody is a laboratory reagent used to detect and purify proteins that have been modified to include a specific HA (hemagglutinin) tag sequence. This antibody can be used in various immunological techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and isolate HA-tagged proteins.
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Lab products found in correlation
Eclipse ti confocal microscope, by Nikon (1 mentions)
Fitc conjugated secondary antibody, by Merck Group (1 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Anti ha tag, by Merck Group (1 mentions)
Anti myh9 11128 1 ap, by Proteintech (1 mentions)
Anti flag tag, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti hsp70, by Cell Signaling Technology (1 mentions)
Anti pdh, by Santa Cruz Biotechnology (1 mentions)
Anti mtch2, by Proteintech (1 mentions)
Anti hsp90, by Cell Signaling Technology (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
F1804, by Merck Group (1 mentions)
P0013, by Beyotime (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Mtt cell viability, by Merck Group (1 mentions)
Streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti ha tag antibody
1
Detect Exogenous HA-Tagged PGRMC1
2
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).
3
Antibody Validation for Organelle Proteins
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The antibodies against Act1, Tom70, and Tom22 were raised in rabbits using recombinant purified proteins. The secondary antibody was obtained from Bio-Rad (Goat Anti-Rabbit IgG [H+L]-HRP Conjugate #172-1019) or ImmunoReagents Inc. (Goat anti-Rabbit IgG [H&L]—Affinity Pure, HRP Conjugate, #GtxRb-003-DHRPX, Goat anti-Mouse IgG [H&L]—Affinity Pure, HRP Conjugate, #GtxMu-003-DHRPX). The horseradish-peroxidase coupled HA antibody was purchased from Roche (Anti-HA-Peroxidase, High Affinity [3F10], #12 013 819 001). The HA-tag antibody used in immunofluorescence and Western blot assays was purchased from Sigma-Aldrich (Anti-HA-Tag antibody produced in rabbits, #SAB4300603). The Tom70 antibody was purchased from Sigma-Aldrich (Anti-TOMM70 antibody produced in rabbit, #HPA048020). For analysis of isolated human mitochondria, the following additional antibodies were used: Anti-Hsp90 (#4874S; Cell Signaling Technology), Anti-PDH (#sc-377092; Santa Cruz), Anti-MTCH2 (#16888-1-AP; Proteintech), Anti-Hsp70 (#4872S; Cell Signaling Technology), Anti-GAPDH (#sc-32233; Santa Cruz), Anti-AK2 (rabbit, selfmade). Antibodies were diluted in 5% (wt/vol) nonfat dry milk in 1× TBS buffer in a 1:1,000 dilution, except for: Anti-HA-Peroxidase 1:500, Anti-Rabbit 1:10,000, and Anti-Mouse 1:5,000.
4
Co-Immunoprecipitation Protocol for HEK293T Cells
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Co-IP was performed as described previously [35 (link)]. Approximately 3.5 × 106 HEK293T cells were harvested ∼24 h after transfection and lysed in 450 µl of cell lysis buffer for Western and IP (Beyotime; P0013). Lysates were precleared by incubating with protein A/G-PLUS agarose beads (Santa Cruz, sc-2003) for 1 h. And the primary antibodies, anti-HA tag antibody (Sigma-Aldrich, H3663) and anti-FLAG tag antibody (Sigma-Aldrich, F1804) were used.
5
11β-HSD1 Overexpression Impacts Cell Viability
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To directly assess the function of 11β-HSD1 in vitro, mouse cDNA with a 3'-(HA)3 tag was sub-cloned into pcDNA3.1 vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA) between the cytomegalovirus promoter and bovine growth hormone polyadenylation sequence and used to transfect murine insulinoma MIN6 cells, which were selected using G418 (Wisent, St-Bruno, QC) for 60 d as reported [5 (link)]. After Western blot confirmation using anti-HA tag antibody (Cat# G036 Abm, Richmond, BC), 11β-HSD1-overexpressing (MIN6-HSD1) and vector transfected (MIN6-Vec) clones were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) cell viability (Sigma-Aldrich) and 5-bromo-2'-deoxyuridine (BrdU) incorporation assays. The cells were cultured in 10% serum for 1 to 3 d in the presence or absence of 11β-HSD1 inhibitor 10j (Cat# 385581, Calbiochem, EMD Millipore, Etobicoke, ON) before the assays were performed. For BrdU incorporation, in the final 18 h of incubation, 10 μM BrdU was added; its incorporation was quantified using ELISA at 450 nm (EMD Millipore).
6
Biotinylation and Internalization Assay of N-Cadherin
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CHO cells were transfected with Ncad-HA(WT) or Ncad GD-HA (GD mutant) and incubated with 1 mg/ml Sulfo-NHS-LC-Biotin(Thermo) in KRPH Buffer (128 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4, 5 mM NaH2PO4, 20 mM HEPES) at 4℃ for 1 hr. Surface-biotinylated cells were washed in PBS and washed twice more with Biotin Blocking Reagent (50 mM NH4Cl, 1 mM MgCl2, 0.1mMCaCl2). Cells were replaced in DMEM/F12 containing 10% FBS and incubated at 37℃ for 1,3,6,12 h. After incubation, cells were lysed in RIPA buffer (20 mM Tris–HCl pH7.4, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, 1% Sodium Deoxycholate, 5 mM EDTA containing a protease inhibitor mix). Surface-biotinylated proteins were pulled down with streptavidin beads (Invitrogen), subjected to SDS PAGE and blotted with anti-HA-tag antibody (Sigma).
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