The Vitreoscilla hemoglobin gene promoter Pvhb was amplified from pET-28a-Pvhb-pelB-asp (Lab store). Meanwhile, the Sumo–Tum 5 fragment was amplified from pSmart-I-Tum 5 (constructed in this study). Pvhb-pelB-SUMO-Tum 5 was obtained by overlap extension PCR and inserted into pET-28a after digestion by Apa I and Xho I. The sequenced vector was transformed into EcN by electroporation and named EcN (Tum-5). pET-28a was also transformed into EcN as negative control and named EcN (28a). EcN (Tum-5), EcN (28a) and EcN were cultured in LB medium for 10 h. Then, Western blot was used to confirm the expression of Tum-5 in both cell supernatant and culture supernatant by using Anti-6×His rabbit polyclonal antibody. The medium supernatant was precipitated with acetone containing 10% TCA and then washed with 90% acetone. Total cell protein (10 μg) was separated through SDS–PAGE and transferred to polyvinylidene difluoride membranes (Millipore) using a semidry method. The result was detected by IRDye® 680 conjugated goat anti-rabbit IgG (LI-COR, America) and observed by Odyssey Infrared Imaging System (LI-COR, America).
Irdye 680 conjugated goat anti rabbit igg
Manufactured by LI COR
Sourced in United States
IRDye 680-conjugated goat anti-rabbit IgG is a secondary antibody used for detection and quantification of rabbit primary antibodies in various immunoassay applications, such as Western blotting and immunohistochemistry. The antibody is conjugated with the IRDye 680 fluorescent dye, which can be detected using near-infrared fluorescence imaging systems.
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Lab products found in correlation
Irdye 800 conjugated goat anti mouse igg, by LI COR (5 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Odyssey system, by LI COR (3 mentions)
Irdye 800cw conjugated goat anti mouse igg, by LI COR (3 mentions)
Pdvf membrane, by Merck Group (2 mentions)
Complete ultra tablet, by Roche (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Odyssey blocking solution, by LI COR (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (2 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Glo lysis buffer, by Promega (1 mentions)
Odyssey v1, by LI COR (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
12 protocols using irdye 680 conjugated goat anti rabbit igg
1
Recombinant Expression of Tum-5 Protein
2
Western Blotting for NeuN and β-Actin
3
Western Blot Analysis of α-SMA and GAPDH
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20 µg protein per lane was separated by 10–12% SDS-PAGE and electro-blotted onto a nitrocellulose membrane, which was probed with anti-α-SMA mouse antibodies (Kangcheng Biological Company, Shanghai, CA) and anti-GAPDH mouse antibodies (Kangcheng Biological Company, Shanghai, CA). IRDye 800CW donkey anti-mouse IgG and IRDye 680 conjugated goat anti-rabbit IgG (LiCor, CA, USA) was used to detect the bound mouse antibodies using an Odyssey test. GAPDH was used as a loading control.
4
Western Blot Protein Detection Protocol
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Samples were solubilized in buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40) containing 0.1% SDS and 4% proteinase inhibitor (complete protease inhibitor cocktail; Roche) and treated with 4× SDS-PAGE loading buffer (final concentration 62.5 mM Tris, pH 6.8, 3% β-mercaptoethanol, 8% SDS, 15% glycerol) at 95°C for 5 min. Afterward, samples were electrophoresed and transferred to a polyvinylidene difluoride membrane (Millipore); the membrane was blocked for 30 min with blocking buffer (5% nonfat dry milk, 50 mM Tris-HCl, pH 7.5, 0.05% Tween-20). After extensive washing with TBS containing 0.1% Tween-20, the immunoblots were incubated with primary antibodies for 2 h. Antibody binding was detected using an IRDye 800–conjugated goat anti-mouse IgG (LI-COR Bioscience) or IRDye 680–conjugated goat anti-rabbit IgG (LI-COR Bioscience) and visualized with an ODYSSEY infrared imaging system (LI-COR Bioscience). For quantification of kininogen immunoblots, the density of immunoblots was calculated using Quantity one software (Bio-Rad); the density of the blots in all film were background-subtracted and reported as adjusted volume (INT × mm2).
5
Antibody Validation and Quantification
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Primary antibodies used in this study include anti-MELK (EPR3981, Abcam or GeneTex), anti-α-tubulin (RRID:AB_1904178 ; 3873S, Cell Signaling Technology), anti-GAPDH (RRID:AB_561053 ; 2118S, Cell Signaling Technology), and anti-Vinculin (RRID:AB_10604160 ; SAB4200080, Sigma). Secondary antibodies used were IRDye680-conjugated goat anti-rabbit IgG (RRID:AB_10956166 ; Cat# 926–68071, LI-COR Biosciences) and IRDye800-conjugated goat anti-mouse IgG (RRID:AB_621842 ; Cat# 926–32210, LI-COR Biosciences).
6
Detection of FXII Cleavage by SDS-PAGE
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Cleavage of FXII into FXIIa was detected by SDS-PAGE (12%) under reducing conditions and immunoblotting. After incubation with apoptotic or viable cells as described above, the cells were pelleted by centrifugation, and samples containing FXII were collected (14 (link)). These samples and samples from a cell-free system were mixed with SDS-PAGE loading buffer and heated at 95°C. After the samples were separated by electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore), the membrane was blocked with 5% non-fat dry milk in blocking buffer. After extensive washing, the immunoblots were incubated for 2 h with the primary antibodies, including monoclonal anti-FXII (B7C9), polyclonal anti-HK heavy chain, and polyclonal anti-pKal antibodies. Antibody binding was detected by IRDye 800-conjugated goat anti-mouse IgG (LI-COR Bioscience) or IRDye 680-conjugated goat anti-rabbit IgG (LI-COR Bioscience) and visualized with the ODYSSEY infrared imaging system (LI-COR).
7
Immunoblot Analysis of p-Smad1/5/8
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Cells were lysed in RIPA cell lysis buffer (Santa Cruz, Santa Cruz, CA) supplemented with protease inhibitor cocktail (Santa Cruz) and phosphatase inhibitor cocktail 2 (Santa Cruz). Protein concentrations were determined by using BCA protein kit (Thermo Fisher), and cell lysate was isolated in SDS-PAGE and transferred onto PVDF membrane. Alpha-tubulin and p-Smad1/5/8 were detected by Odyssey system (Li-Cor bioscience) following incubation with the appropriate primary and secondary antibodies. Primary antibodies used were rabbit anti-p-Smad1/5/8 (Cell Signaling Tech, 1∶1000 dilution) and mouse anti-alpha-tubulin (Cell Signaling Tech, 1∶1000 dilution). The secondary antibodies used include IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience, 1∶5000 dilution) and IRDye 800CW-conjugated goat anti-mouse IgG (Li-Cor Bioscience, 1∶5000 dilution).
8
Immunoprecipitation of SR-BI in Transfected Cells
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Untransfected YB 2/0 cells and SR-BI transfected YB 2/0 cells were reacted with serum with anti-SR-BI activity or control serum. The cells were lysed with M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology Inc., Rockford, IL, USA) followed by incubation with 25 µL of Protein A/G Magnetic Beads (Pierce Biotechnology Inc.). After washing with Tris-buffered saline [10 mM tris (pH 7.5), 150 mM NaCl] containing 0.1% Tween 20, eluted with 2× Laemmli sample buffer (Bio-Rad Laboratories). Under non-reducing conditions, Western blotting was performed as described above. The membranes were treated with rabbit anti-SR-BI antibody (NB400-104, Novus Biologicals, 1:1000), IRDye 680-conjugated goat anti-rabbit IgG (926-32221, LI-COR Biosciences, 1:5000), or IRDye 800CW-conjugated goat anti-human IgG (H + L) (926-32232, LI-COR Biosciences, 1:5000).
9
Western Blot Analysis of Smad Signaling
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Cells were lysed with RIPA buffer (Sigma) containing protein inhibitors (complete ULTRA Tablets, Roche, Basel, Switzerland) and phosphatase inhibitors (PhosSTOP, Roche, Basel, Switzerland). Samples were denatured by incubating at 95 °C for 5 min in sample buffer and separated by using SDS-PAGE (precast 8–16% gradient gels, Biorad, Hercules, CA, USA). Then, the samples were transferred to a PDVF membrane (Millipore). The membrane was blocked with Odyssey Blocking solution (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature, followed by primary antibody incubation at 4 °C overnight. The primary antibodies used in the present study included rabbit anti-p-Smad1/5/9 (Cell Signaling Tech, Danvers, MA, USA), rabbit anti-Smad 1(Cell Signaling Tech), mouse anti-beta actin (Cell Signaling Tech), rabbit anti-p-Smad 2 (Cell Signaling Tech) and rabbit anti-Smad 2 (Cell Signaling Tech). The proteins were detected by Odyssey system (Li-Cor bioscience) followed by the secondary antibodies including IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience) and IRDye 800CWconjugated goat anti-mouse IgG (Li-Cor Bioscience, Lincoln, NE, USA).
10
Protein Extraction and Western Blot
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