Protein samples were incubated at 70°C for 10 min after adding 3× SDS sample buffer (stock concentration 30% glycerol, 3% SDS, 93.75 mM Tris‐Cl pH 6.8, 0.06% bromophenol blue). These samples were loaded on SDS–PAGE gels (8 or 12%) and run at 90 V. After dye front reached the end, these gels were transferred with TransBlot (Biorad) at conditions of 1.0 mA for 30 min onto PVDF membranes. Transferred membranes were blocked with 5% skim milk in TBST, and antibodies were added subsequently and incubated overnight at 4°C. The following antibodies were used; Flag‐HRP (Sigma, A8592), Myc‐HRP (Sigma, 16‐213), V5‐HRP (Sigma, V2260), HA‐HRP (Roche, 12013819001), MPK6 (Agrisera, AS12 2633), H+‐ATPase (Agrisera, AS07 260), and GFP‐HRP (Abcam, ab6663). Signals were detected using ECL substrates (Thermo Fisher, 34580). After detection, membranes were stained with Ponceau S solution (Sigma, P7170) to use as loading control. PageRuler™ Prestained Protein Ladder (Thermo Scientific, 26616) was used as molecular weight markers.
Flag hrp
Manufactured by Merck Group
Sourced in United States
Flag-HRP is a laboratory reagent that consists of a horseradish peroxidase (HRP) enzyme fused to a FLAG peptide tag. The FLAG tag allows for the detection and purification of proteins that have been expressed with the FLAG-HRP fusion. The HRP enzyme can be used as a reporter in various immunoassays and other biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ha hrp, by Roche (4 mentions)
Gst hrp, by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Ha hrp, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
β actin, by Merck Group (3 mentions)
V5 hrp, by Merck Group (2 mentions)
γ tubulin, by Merck Group (2 mentions)
Ccnd2, by Cell Signaling Technology (2 mentions)
Cdkn1b, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by PerkinElmer (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Baf170, by Fortis Life Sciences (2 mentions)
T7 antibody, by Merck Group (2 mentions)
T7 hrp, by Merck Group (2 mentions)
Baf155, by Cell Signaling Technology (2 mentions)
Gapdh hrp, by Cell Signaling Technology (2 mentions)
Ha epitope tag, by Cell Signaling Technology (2 mentions)
Gapdh, by Merck Group (2 mentions)
41 protocols using flag hrp
1
Immunoblotting of Tagged Proteins
2
Comprehensive Protein Detection Techniques
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For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, ♯sc-47778; 1:5000), ATF6 (Cell Signaling Technology, ♯65880; 1:1000), BCL-2 (AbCam, ♯ab182858; 1:500), BCL-XL (Cell Signaling Technology, ♯2764; 1:1000), Cleaved-Caspase-8 (Cell Signaling Technology, ♯8592; 1:1000), Caspase-3 (Cell Signaling Technology, ♯9665; 1:1000), Caspase-9 (Cell Signaling Technology, ♯9508; 1:1000), CHOP (Cell Signaling Technology, ♯2895; 1:250), GFP (AbCam, ♯ab13970; 1:2000), GRP78 (Cell Signaling Technology, ♯3177; 1:1000), FLAG-HRP (Sigma Aldrich, ♯A8592; 1:4000), HA-HRP (Roche, ♯11867423001; 1:2000), IP3R1 (Thermo Fischer, ♯PA1-901; 1:1000), IP3R1 & IP3R2 [previously described77 ; 1:1000], IP3R3 (BD Biosciences, ♯610312; 1:1000), KDEL (AbCam, ♯ab12223; 1:2000), MCL-1 (Cell Signaling Technology, ♯5453; 1:1000), Nicastrin (BD Biosciences, ♯612290; 1:1000), PARP (Cell Signaling Technology, ♯9542; 1:1000), SERCA2 (Cell Signaling Technology, ♯4388; 1:1000), STIM1 (Cell Signaling Technology, ♯5668; 1:1000).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP3R1 (Thermo Fischer, ♯PA1-901; 1:200), IP3R3 (BD Biosciences, ♯610312; 1:200).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP3R1 (Thermo Fischer, ♯PA1-901; 1:200), IP3R3 (BD Biosciences, ♯610312; 1:200).
3
Western Blotting Antibody Deployment
4
Immunoblotting of hASIC1 Trimers
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Cells expressing hASIC1 trimers were homogenized with buffer containing (mM): 150 NaCl, 50 Tris-base ph 7.4, 5 EDA, 1% Triton X-100 on ice for 15 min. Lysates were cleared by centrifugation 10 K rpm at 4°C. After protein quantification (Pierce BCA Protein Assay Kit), 30 µg of protein were loaded on SDS-10% polyacrylamide gels. Proteins were transferred to membranes (Immobilon-P CN; IPVH00010, Millipore) by electrophoresis and subsequently processed for blotting with HA (Santa Cruz Biotechnologies), Flag-HRP (SigmaAldrich) or V5-HRP (ThermoFisher Scientific) mouse monoclonal antibodies. After washes, the blot using HA monoclonal received anti-mouse secondary antibody labeled with HRP (ThermoFisher Scientific).
5
Western Blot Analysis of Chromatin Remodelers
6
Western Blot Analysis of Signaling Proteins
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Cells were washed with PBS and lysed in extraction buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF and protease inhibitor cocktails]. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore corporation, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBS-T [10 mM Tri-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and immunoblotted with antibodies against c-Fos (Santa Cruz Biotechnology, Dallas, TX, USA), NFATc1 (Santa Cruz Biotechnology), Phospho-STAT5 (Cell Signaling Technology), STAT5A (Cell Signaling Technology, Beverly, MA), IκB (Cell Signaling Technology), Flag-HRP (Sigma-Aldrich), Actin-HRP (Sigma-Aldrich), mouse-IgG-HRP (Abcam, Cambridge, UK) and rabbit-IgG-HRP (Abcam). Signals were detected with ECL solution (Millipore corporation) and analyzed using a LAS3000 luminescent image analyzer (GE Healthcare, Piscataway, NJ, USA).
7
Protein Extraction and Immunoblotting Protocol
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Protein extracts were obtained by grinding frozen tissues in liquid nitrogen. After resuspension in 2X Laemmli Buffer, the extracts were treated for 5 min at 95°C and centrifuged before loading on SDS/PAGE gels. Samples were electroblotted to PVDF membrane (Immobilon, Millipore) and proteins of interest were visualized using the antibodies described in the text. Antibodies working concentrations were as follow: HA-HRP 1:10000 (H6533 Sigma), Cmyc 1:40000 (sc-789 Santa-Cruz), Flag-HRP 1:7500 (A8592 Sigma), GFP 1:2000 (632592 Clonetech), UGPase 1:10000 (AS05086 Agrisera), H3 1:30000 (07–690 Millipore), DCL1 1:1000, DCL3 1:1000, DCL4 1:500, RDR2 1:5000, AGO4 1:12000. All hybridization were performed in 1X TBS, 0.5% Tween, 5% milk overnight at 4°C.
8
Western Blot Analysis of Muscle Markers
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Tissue samples and cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). After adding SDS loading buffer, samples were boiled for 10 min. Protein samples were resolved by SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked and incubated with primary antibodies. Antibodies used were TY1 (Sigma, SAB4800032), HRP2 (Proteintech, 15134-1-AP), MHC (Developmental Studies Hybridoma Bank, MF20), Myog (Santa Cruz, SC-12732), GAPDH (Millipore, MAB347), FLAG-HRP (Sigma, F7425), DPF3a (Custom made), P-DPF3a (Custom made), Pan-DPF3a (Custom made), GST (Santa Cruz, sc-459) and MYOD (Santa Cruz, SC-304).
9
Immunodetection of FLAG-tagged Proteins
10
Antibody Dilution Protocol for Immunoblotting
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FLAG-HRP (Sigma-Aldrich, A8592) used at 1:2000 dilution in Fig. 1c , Supplementary Fig. 1b , and Supplementary Fig. 1c . Sam68 (KHRDBS1) polyclonal antibody (Sigma-Aldrich, S9575) used at 1:1000 dilution in Supplementary Fig. 1c . Tubulin monoclonal antibody (Sigma-Aldrich, T9026) used at 1:5000 dilution in Supplementary Fig. 1c .
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