Hes1 mRNA was quantitated by smFISH, as previously described (9 ), using a set of 32 × 20-nt probes against Human Hes1 exonic sequence, labeled with Quasar 570 (Biosearch Technologies) at their 5' end. Immunostainings were performed following standard protocols using either commercial HES1 primary antibody (E5 clone, sc-166410, Santa Cruz Biotechnology, on formaldehyde-fixed MCF-7 cells) or mouse anti-PCNA (M0879, Dako) for cell-cycle phases estimation, after smFISH protocol. For protein half-life estimation of exogenous HA-Hes1, MCF-7 cells transfected with pCS2-HA-Hes1 plasmid and treated with 100 uM CHX for the time points indicated and analyzed by Western blot using a using HA-HRP antibody (Roche).
Ha hrp antibody
Manufactured by Roche
The HA-HRP antibody is a reagent used in various laboratory techniques. It is a horseradish peroxidase (HRP) conjugated antibody that specifically binds to the hemagglutinin (HA) tag, which is a commonly used protein tag. The HA-HRP antibody can be used to detect and quantify HA-tagged proteins in Western blotting, ELISA, and other immunoassays.
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Lab products found in correlation
Quasar 570, by Biosearch Technologies (1 mentions)
M0879, by Agilent Technologies (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Pcmv gag pol, by Cell Biolabs (1 mentions)
Anti flag m2 horse radish peroxidase antibody, by Merck Group (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
Pbabe puro, by Cell Biolabs (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ezview red anti ha affinity gel, by Merck Group (1 mentions)
Protein a g agarose, by Roche (1 mentions)
Anti bcl2 sc 509, by Santa Cruz Biotechnology (1 mentions)
Anti ha matrix, by Roche (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ha hrp antibody
1
Quantifying Hes1 mRNA and Protein Dynamics
2
Generation and Purification of PAWS1 Antibodies
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A PAWS1 antibody was generated by injecting GST-PAWS1 (amino acids 715–815) into sheep. The P-PAWS1 S610 antibody was generated by injecting peptide GPGPRRPS*VAS (* denotes phospho-Ser) into rabbit. The antibodies were subsequently affinity purified. Anti-FLAG-M2-horseradish peroxidase (HRP) antibody was from Sigma. HA-HRP antibody was from Roche. Antibodies recognizing phospho-SMAD1/5/8, phospho-SMAD2, phospho-SMAD3, SMAD2/3, GAPDH and lamin A/C were from Cell Signalling Technology. HRP-conjugated secondary antibodies and light-chain-specific HRP-conjugated antibodies were from Jackson Laboratories. BMP-2 and BMP-4/7 were from R&D Systems. The nuclear cytoplasmic extraction kit was from Thermo Scientific. The first strand cDNA synthesis kit was from Invitrogen. 2X SYBR green master mix was from BioRad. pBABE-Puro, pCMV-Gag-Pol and pCMV-VSVG constructs were from Cell Biolabs. All plasmids for mammalian cell expression were cloned into pCMV5, pBABE-Puro or pcDNA-FRT-TO vectors with N-terminal FLAG, HA or GFP tags as indicated. For bacterial expression of proteins, SMAD1, SMAD2 and PAWS1 (523-end; other mutants) were cloned into pGEX6T vectors. All DNA constructs were verified by DNA sequencing (by the DNA Sequencing Service at University of Dundee; www.dnaseq.co.uk ). Bacterial protein expression in BL21 cells and purification were performed as described previously [31 (link)].
3
SARS-CoV-2 Spike Glycoprotein Purification
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When indicated, a secreted form of doubly tagged spike-glycoprotein of SARS-CoV-2 (N-terminal HA tag and C-terminal V5 tag) from media of cocultured HeLa and HeLa-TZM-bl cells was analyzed by immunoprecipitation. Namely, 0.3 mL of media were precipitated with 25 μL of EZ view red anti-HA affinity gel (E 6779; Sigma-Aldrich) according to the manufacturer’s protocol. Upon SDS-PAGE separation and PVDF transfer, the proteins were detected using an HA-HRP antibody (12-013-819; 1:3,500; Roche).
4
Casp8p41-HA Expression and Protein Interactions
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Activated CD4 cells (as mentioned above) were transfected with Casp8p41-HA expression vector using Lanza Nucleofector IIb system. One hour after electroporation, cells were treated with Z-VAD-fmk (10 μM) and Ixazomib (100 nM) for 4 h (to prevent protein degradation and cell death induced by Casp8p41 expression), followed by treatment with or without bryostatin (10 ng/ml) for an additional 1 h. Cells were collected and washed and lysis with cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1.0% Tween-20, protease and phosphatases inhibitors). Five hundred micrograms of total cell lysate were diluted in 500 μl of buffer, precleared with 25 μl Protein A/G agarose (Santa Cruz). Precleared lysate incubated with Anti-HA matrix (cat # 11815016001, Roche, Germany) or 2 μg anti–BCL2 (SC-509, Santa Cruz) or mouse IgG for overnight at 4C. After incubation, 20 μl of Protein A/G agarose was added to the appropriated samples, and complexes were washed five times in IP buffer (20 mM Tris, pH 7.5, 300 mM NaCl, 1.0% Tween-20, protease and phosphatases inhibitors) and boiled in 20 μl 2× SDS-PAGE loading buffer, subjected to SDS-PAGE, and immunoblotted with HA-HRP antibody (Roche) or BCL2 antibody.
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