Anti ha probe f 7

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The Anti-HA probe F-7 is a monoclonal antibody that specifically recognizes the hemagglutinin (HA) epitope tag. It is a useful tool for the detection and immunoprecipitation of HA-tagged proteins in various experimental applications.

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6 protocols using anti ha probe f 7

ChIP-qPCR Assay for Chromatin Immunoprecipitation

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ChIP‐qPCR was performed as described in Poli et al (2016 (link)) using anti‐HA probe F‐7 (Santa Cruz, sc‐7392) and anti‐Rpb1‐CTD 8WG16 (Abcam, ab817) coupled to Dynabeads (Invitrogen, protein A and sheep anti‐mouse M280 IgG). For quantitative PCR, background controls were determined using uncoupled Dynabeads and enrichment was normalized to chromatin Input. Primers used for ChIP‐qPCR are listed in Table EV2.

Hurst V., Challa K., Jonas F., Forey R., Sack R., Seebacher J., Schmid C.D., Barkai N., Shimada K., Gasser S.M, & Poli J. (2021). A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress. The EMBO Journal, 40(21), e108439.

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Molecular Interplay of Ubiquitin Proteases

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bAP15 (11324) was purchased from Cayman Chemical (Ann Arbor, MI). The USP14 inhibitor IU1 (662210) was obtained from EMD Millipore Corporation (Billerica, MA). UCHL5 shRNA plasmid (h) (sc-76797-SH) was purchased from Santa Cruz Biotechnology (Dallas, TX). HA-ubiquitin (Plasmid 18712) was purchased from Addgene (Cambridge, MA).
Antibodies were obtained as follows; anti-β-actin (sc-47778), anti-UCHL5 (sc-271002), anti-USP14 (sc-100630), anti-β-catenin (sc-7963), anti-Vimentin (sc-6260), anti-Ub (sc-166553), and anti-HA-probe (F-7) (sc-7392) (Santa Cruz Biotechnology), anti-E-cadherin (610181), anti-N-cadherin (610920)(BD Bioscience, San Jose, CA), anti-Smad2 (5339), anti-Phospho-Smad2 (3108), anti-Phospho-Smad2 (18338), anti-Smad3 (9523), anti-Phospho-Smad3 (9520), and anti-Smad4 (38454) (Cell Signaling Technology, Danvers, MA).

Fukui S., Nagasaka K., Miyagawa Y., Kikuchi-Koike R., Kawata Y., Kanda R., Ichinose T., Sugihara T., Hiraike H., Wada-Hiraike O., Sasajima Y, & Ayabe T. (2019). The proteasome deubiquitinase inhibitor bAP15 downregulates TGF-β/Smad signaling and induces apoptosis via UCHL5 inhibition in ovarian cancer. Oncotarget, 10(57), 5932-5948.

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Immunoprecipitation and Western Blotting

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The cells were rinsed twice in ice-cold HBSS and were lysed in ice-cold immunoprecipitation buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4, 1 mmol/L EDTA, 1 mmol/L ethylene glycol bis [β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, pH 8.0, 0.2 mmol/L sodium orthovanadate, and protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)]. The samples were prepared as previously described (36 (link)). The blots were probed with anti-HA-probe (F-7, 1:1,000, SC-7392, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-c-myc (PE10, SC-40, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies.

Jiao Y., Zhang Y.G., Lin Z., Lu R., Xia Y., Meng C., Pan Z., Xu X., Jiao X, & Sun J. (2020). Salmonella Enteritidis Effector AvrA Suppresses Autophagy by Reducing Beclin-1 Protein. Frontiers in Immunology, 11, 686.

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Antibody Detection Protocol for Cell Signaling

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Antibodies to cyclin F (D9K2U), USP7 (D17C6), UBR5 (D6O8z), LC3 (4775S), and β-Actin (13E5) were from Cell Signaling Technology (MA, USA). Anti-HA-probe (F7) was from Santa Cruz (TX, USA), anti-cyclin A2 (BF683) from Bethyl Laboratories (TX, USA), anti-p53 (DO7) from Abcam (Cambridge, UK), anti-rabbit and anti-mouse IgG-HRP from Genei Laboratories (Bangalore, India), and anti-rabbit IgG-HRP from Cloud-Clone Corp (TX, USA).

Sharma S.S., Pledger W.J, & Kondaiah P. (2022). The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability. Aging (Albany NY), 14(21), 8645-8660.

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Immunofluorescent Detection of HA and Phospho-CDK4

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Cells were fixed in 4% paraformaldehyde for 10 min at 4°C, then methanol for 10 min at −20°C, and permeabilized with 0.1% Triton X-100 before immunofluorescent detection. Anti-HA probe (F7, Santa Cruz Biotechnology) or anti-phospho-CDK4 (T172) monoclonal (NB8-AD9) antibodies were followed by a biotinylated anti-mouse immunoglobulin antibody (GE Healthcare) and Alexa Fluor 488-conjugated streptavidin (Jackson Immunoresearch).

Coulonval K., Vercruysse V., Paternot S., Pita J.M., Corman R., Raspé E, & Roger P.P. (2021). Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27. Cell Cycle, 21(1), 12-32.

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Western Blot Analysis of Protein Targets

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A total of 40 μg protein extract was boiled for 10 min in SDS sample buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting as reported in Di Sanzo et al. (38 (link)). The nitrocellulose membranes were incubated overnight at 4°C with the following antibodies: (a) anti-CXCR4 (1:500; Abcam), (b) anti-HIF-1α (H-206) (1:200; Santa Cruz Biotechnology), (c) anti-p65 (C-20) (sc-372, 1:1,000; Santa Cruz Biotechnology), (d) anti-HDAC (1:5,000; Sigma-Aldrich), (e) anti-HA probe (F-7) (1:1,000; Santa Cruz Biotechnology), (f) anti-Vimentin, (g) anti-E-cadherin, (h) anti-Snail, (i) anti-Slug (1:1,000; Cell Signaling Technology, Danvers, MA, USA), (l) anti-FtH (1:200; Santa Cruz Biotechnology), (m) anti-γ-Tubulin (C-20) (1:2,000; Santa Cruz Biotechnology), (n) anti-Nucleolin (D4C7O) (1:1,000; Cell Signaling Technology) over-night at 4°C, followed by incubation with goat anti-rabbit and mouse anti-goat secondary antibodies (1:5,000; Santa Cruz Biotechnology). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactive bands were visualized with the ECL Western blotting detection system (BioRad, Hercules, CA, USA).

Chirillo R., Aversa I., Di Vito A., Salatino A., Battaglia A.M., Sacco A., Di Sanzo M.A., Faniello M.C., Quaresima B., Palmieri C., Biamonte F, & Costanzo F. (2020). FtH-Mediated ROS Dysregulation Promotes CXCL12/CXCR4 Axis Activation and EMT-Like Trans-Differentiation in Erythroleukemia K562 Cells. Frontiers in Oncology, 10, 698.

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