Following rinsing in ice-cold PBS, cells were lysed in radioimmunoprecipitation assay buffer containing 1% protease inhibitor (Thermo Fisher Scientific, Inc.), protein concentration was detected by NanoDrop. A total of 50 µg protein per lane was separated using 10% SDS-PAGE. The proteins were transferred to nitrocellulose membranes (Thermo Fisher Scientific, Inc.), which were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk (BD Biosciences) for 2 h at room temperature. The membranes were sequentially incubated with the appropriate primary antibodies against LASP1 (1:1,000; cat. no. ab156872; Abcam) overnight at 4°C by gentle shaking and subsequently with Alexa Fluor® 647-conjugated goat anti-rabbit secondary antibodies (1:1,000; cat. no. 4414; Cell Signaling Technology, Inc.) for 1 h at room temperature. The Odyssey fluorescence scanning system (LI-COR Biosciences) and Image Studio software (V4.0; LI-COR Biosciences) were used to detect immunoreactivity. GAPDH (1:1,000; cat. no. ab9485; Abcam) was used as a loading control.
Alexa fluor 647 conjugated goat anti rabbit secondary antibodies
Manufactured by Abcam
Sourced in United States
Alexa Fluor® 647-conjugated goat anti-rabbit secondary antibodies are fluorescently-labeled secondary antibodies that bind to rabbit primary antibodies. The Alexa Fluor® 647 dye provides a far-red fluorescent signal.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Odyssey fluorescence scanning system, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Nonfat milk, by BD (1 mentions)
C2 confocal microscope, by Nikon (1 mentions)
2 protocols using alexa fluor 647 conjugated goat anti rabbit secondary antibodies
1
Western Blot Analysis of LASP1 Protein
2
NFAT Nuclear Translocation Assay
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Nuclear translocation of NFAT was investigated using the laser scanning confocal microscopy. T lymphocytes were fixed with formalin. After washing, the cells were incubated with 5% goat serum with 0.2%Triton-X100 at room temperature for 1 h. The cells were then treated with anti-NFAT primary antibody (1:50 dilution; Abcam, USA) at room temperature for 4 h. After washing, the cells were incubated with corresponding Alexa Fluor 647-conjugated goat anti-rabbit secondary antibodies (1:200 dilution; Abcam) at room temperature for 2 h. After staining with DAPI for 30 min and washing with PBS, the fluorescence in the cells was observed with the C2+ Confocal Microscope (Nikon).
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