Cells were fixed for 30 min at room temperature using 4% paraformaldehyde solution. This was followed by permeabilization using 0.25% Triton X-100 for 15 min. Blocking was performed by using 1% BSA/0.2% Tween 20 solution for 30 min. The cells were then treated with the anti-FLAG antibody (1:500, Sigma, F1804) or anti-HA probe (1:500, (F-7) sc-7392, Santa Cruz) followed by the Alexa 488 conjugated anti-mouse secondary antibody (Thermofisher) and counter-stained with Hoechst 33342 (Thermofisher). A Zeiss LSM700 confocal microscope was used for capturing the images.
Anti ha probe
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-HA-probe is a monoclonal antibody that specifically recognizes the hemagglutinin (HA) epitope tag. It is commonly used in research applications to detect and immunoprecipitate HA-tagged recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx scanner, by LI COR (2 mentions)
Anti actin, by Merck Group (2 mentions)
Irdye 680rd donkey anti mouse, by LI COR (2 mentions)
Irdye 680rd goat anti rabbit, by LI COR (2 mentions)
Donkey irdye800cw anti mouse, by LI COR (2 mentions)
Goat α rabbit irdye 800cw, by LI COR (2 mentions)
Image studio software, by LI COR (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Alexa 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti ub, by Santa Cruz Biotechnology (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
Anti octa probe, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Ez western lumi pico, by Daeil Lab Service (1 mentions)
Nitrocellulose membrane, by Macherey-Nagel (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
9 protocols using anti ha probe
1
Immunofluorescence Staining of Cells
2
Western Blot Quantification Protocol
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Immunoblots were performed as described 74 (link). Samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked in 50 mM Tris-buffered saline with 0.1% Tween-20 (TBST, pH 8.0) containing 4% nonfat milk for 1 hr at room temperature and probed with primary antibodies: 1) anti-HA-probe (Santa Cruz Biotechnology, Cat# sc-7392, 1:500), 2) anti-Ub (Santa Cruz Biotechnology, Cat# sc-8017) or anti-actin (Sigma-Aldrich, Cat# A2066, 1:1,000) in TBST containing 2% BSA at 4°C overnight. After washing 3X with TBST, the membranes were incubated with secondary antibodies: donkey IRDye 680RD anti-mouse (LI-COR Biosciences, Cat# 926–68072, 1:20,000), Goat IRDye 680RD anti-rabbit (LI-COR Biosciences, Cat# 926–68071, 1:20,000), and/or donkey IRDye800CW anti-mouse (LI-COR Biosciences, Cat# 926–32212, 1:20,000), goat IRDye 800CW α-rabbit (LI-COR Biosciences, Cat# 926–32211, 1:20,000) for 45 min at room temperature. Capture and analysis were performed using Li-Cor Odyssey CLX scanner and Image Studio software (LI-COR Biosciences).
3
Immunoblot Analysis of Ubiquitinated Proteins
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Immunoblots were performed as described 77 (link). Samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked in 50 mM Tris-buffered saline with 0.1% Tween-20 (TBST, pH 8.0) containing 4% nonfat milk for 1 hr at room temperature and probed with primary antibodies: 1) anti-HA-probe (Santa Cruz Biotechnology, Cat# sc-7392, 1:500), 2) anti-Ub (FK1; Santa Cruz Biotechnology, Cat# sc-8017) or anti-actin (Sigma-Aldrich, Cat# A2066, 1:1,000) in TBST containing 2% BSA at 4°C overnight. After washing 3X with TBST, the membranes were incubated with secondary antibodies: donkey IRDye 680RD anti-mouse (LI-COR Biosciences, Cat# 926–68072, 1:20,000), Goat IRDye 680RD anti-rabbit (LI-COR Biosciences, Cat# 926–68071, 1:20,000), and/or donkey IRDye800CW anti-mouse (LI-COR Biosciences, Cat# 926–32212, 1:20,000), goat IRDye 800CW α-rabbit (LI-COR Biosciences, Cat# 926–32211, 1:20,000) for 45 min at room temperature. Capture and analysis were performed using Li-Cor Odyssey CLX scanner and Image Studio software (LI-COR Biosciences).
4
Western Blot Analysis of Melanogenic Proteins
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The cells were harvested and lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitors. The lysates were incubated for 20 min on ice, further sonicated, and centrifuged at 4 °C. The supernatants were collected and measured by using the BCA Kit (Pierce, Appleton, WI, USA). Equal amounts of protein samples were separated via SDS-PAGE and then, transferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk for 1 h and incubated with primary antibodies against SMILE (NBP2-94859, Novus Biologicals, Littleton, CO, USA), MITF (MA5-14146, Thermo Fisher Scientific), p-CREB (#9198), CREB (#9197), GAPDH (#2118, Cell Signaling Technology, Danvers, MA, USA), β-actin (A5441, Sigma-Aldrich, Burlington, MA, USA), tyrosinase (sc-7833), TRP1 (sc-10443), TRP2 (sc-10451), anti-OctA probe (sc-166355), and anti-HA probe (sc-7392, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with secondary antibodies (Thermo Fisher Scientific). The blots were developed using an EZ-Western Lumi Pico or Femto Western blot detection kit (Daeil Lab Service, Seoul, Republic of Korea).
5
Western Blot Analysis of Protein Targets
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Proteins were extracted using Tris-HCl buffer 0.1 M pH 7.5 that contained 1 mM EDTA, 150 mM NaCl, 0.1% NP40, 0.1 mM Na orthovanadate, 2 mM DTT, and 1 mM PMSF (lysis buffer), separated by SDS-PAGE, and transferred to nitrocellulose membranes (Macherey Nagel), which were then incubated with 5% non-fat milk in PBS-1% Tween. Primary antibody was added at the appropriate dilution and membranes were incubated for 16 h at 4°C. Primary antibodies used were: (1) rabbit polyclonal antibody anti-PKC-alpha (Cell Signaling Technology, 1/1000); (2) rabbit polyclonal antibody anti-Rac(1/2/3) (Cell Signaling Technology, 1/1000); (3) mouse monoclonal anti-c-Myc antibody (Santa Cruz Biotechnologies, 1/1000); (4) rabbit polyclonal antibody anti-HA-probe (Santa Cruz, 1/1000); (5) mouse monoclonal anti-alpha-actin (Millipore, 1/2000); (6) rabbit polyclonal antibody anti-LC3 (Sigma-Aldrich, 1/1000); (7) rabbit polyclonal antibody anti-GFP (Invitrogen, 1/1000); isotype-specific secondary antibody coupled with a horseradish peroxidase (Pierce, 1/2000) was detected by incubating with ECL+ (GE Healthcare) and visualized with CCD camera (FUJI Las 4000).
6
Detecting Sirt1 Ubiquitination in SW480 Cells
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Antibodies include anti-Sirt1 (Santa Cruz, polyclonal sc-15404), anti-HA-probe (Santa Cruz, polyclonal sc-805), anti-Myc (Proteintech Group, Inc., 10828-1-AP), anti-GFP (GeneTex, Inc., Monoclonal GT859), anti-Ubc13 (Cell Signaling Technology, Monoclonal #6999), and anti-IgG (Santa Cruz, polyclonal sc-66931). SW480 cells were lysed in Tris/HCl, pH 7.5, buffered with 1% Triton containing protease inhibitors as described above. Supernatant was incubated with appropriate antibody (2 μg) for at least 90 min at 4 °C followed by incubation overnight with Protein A/G-Sepharose beads (GE Healthcare). After overnight incubation, the agarose beads were washed four times with cold lysis buffer, incubated for 10 min at 108 °C with loading buffer, and subjected to SDS-PAGE and western blot analysis. To detect Sirt1 ubiquitination, 10 mM N-ethylmaleimide was included in the lysis buffer containing a protease inhibitor cocktail (Roche, Hongkong, China).
7
Immunofluorescence Staining of Neural Markers
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Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Samples were blocked with 0.1% Triton X-100 (Sigma-Aldrich) and 5% donkey serum in PBS for 30 min and were subsequently incubated with the primary antibodies: anti-NESTIN (1/200; Merck-Millipore, Burlington, MA, USA ABD69), anti-PAX6 (1/100; DSHB, Douglas, Houston, TX, USA AB 528427), anti-α-Synuclein (αSYN; 1/500; BD Biosciences, Franklin lakes, NJ, USA 610787), anti-βIII-tubulin (TUJ1, 1/1000; Cell Signaling, Danvers, MA, USA 5568), anti-Synapsin 1 (1:200; Cell Signaling), and anti-HA-probe (1:500; Santa Cruz, Dallas, TX, USA) at 4 °C overnight, followed by incubation with appropriate secondary antibodies (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) conjugated to AlexaFluor 488 (green) or 546 (red), for at least 1 h at room temperature. Coverslips were mounted with ProLong Gold antifade reagent with DAPI (Cell Signaling) and images were acquired using a Leica TCS-SP8 confocal microscope (LEICA Microsystems) and analyzed using ImageJ software (version 1.53c) (NIH).
8
Quantifying Protein Expression by Western Blotting
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Western blotting techniques were modified from previous methods [29 (link), 59 (link)]. Proteins tagged with the HA epitope (Snf1, Glc7) were detected with Anti-HA probe (Santa Cruz) diluted 1:2,000. Goat anti-mouse IRDye 800CW (Li-Cor) diluted 1:5,000 was used as the secondary antibody. For detection of phosphorylated Snf1, Phospho-AMPKalpha (Thr172) antibody (Cell Signaling) diluted 1:1,000 was used. Goat anti-rabbit IRDye 800CW (Li-Cor) was used as the secondary antibody at a 1:10,000 dilution. Proteins tagged with the V5 epitope (Hxk2, Reg1) were detected with the Anti-V5 probe (Invitrogen) diluted 1:1,000. Goat anti-mouse IRDye 680 (Thermo) diluted 1:10,000 was used as the secondary antibody. Blots were visualized using an Odyssey Infrared Imager (Li-Cor), and band quantification was performed using Odyssey software.
9
Immunofluorescence Assay for Protein Localization
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Immunofluorescence assay was performed as described previously (Yoo et al., 2009 (link); 2014 (link)). In brief, HeLa cells were grown on glass coverslips coated in poly-D-lysine (0.1 mg/ml, Sigma) and extracted in Triton X-100 buffer (0.5% Triton X-100, 20 mM HEPES-KOH pH 7.4, 50 mM NaCl, 3 mM MgCl2 and 300 mM sucrose). Fixation was then carried out with 4% paraformalde-hyde/2% sucrose in PBS for 30 min, after which the cells were permeabilized with Triton X-100 buffer for 20 min at RT. After blocking with 5% BSA in PBS for 1 h, cells were incubated with anti-PinX1 (1:100, Abnova), anti-HA-probe (1:200, Santa Cruz) or anti-Fibrillarin (1:400, Abcam) at 4°C overnight. Cells were then washed with cold PBS and incubated with Alexa Fluor® anti-mouse or rabbit IgG-488-green (1:500, Invitrogen), Alexa Fluor® anti-rabbit IgG-633-red (1:500, Invitrogen) for 1 h at RT (keep in dark) before incubating with 1.0 μg/ml of DAPI (1.0 mg/ml) (Sigma) for DNA staining. Coverslips were mounted with ProLong® Gold anti-fade reagent (Invitrogen) and sealed with nail polish. Images were acquired by confocal microscopy (Leica, Germany) using the operating software with the Leica Application Suite AF Lite system (Leica).
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