Samples separated by SDS-PAGE (with 20 μg of loaded TMV) were transferred to nitrocellulose membranes at 100 V for 1 hour. Membranes were incubated in 5% w/v milk in TBS-Tween at RT for 1 hour, followed by incubation with either 0.5 μg/mL rabbit anti-TMV antibody (Pacific Immunology) or 0.5 μg/mL rabbit polyclonal anti-albumin antibody (Novus Biologicals) in 5% w/v milk in TBS-Tween for 1 hour at RT. Samples were then washed three times for 10 min each in TBS-Tween and incubated in 1 μg/mL alkaline phosphatase goat anti-rabbit antibody in 5% w/v milk in TBS-Tween at RT for 1 hour, followed by three washes for 10 min each in TBS-Tween and 1 min wash in Millipore water. Antibody binding was visualized using Novex AP Chromogenic Substrate (BCIP/NBT; Invitrogen) (Fig. 1C ).
Novex ap chromogenic substrate bcip nbt
Manufactured by Thermo Fisher Scientific
Sourced in United States
Novex® AP Chromogenic Substrate (BCIP/NBT) is a laboratory reagent used for the detection and visualization of alkaline phosphatase (AP) activity in various applications, such as Western blotting, ELISA, and immunohistochemistry. The substrate consists of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT), which react with the AP enzyme to produce a purple-blue colored precipitate, indicating the presence and location of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Anti sod1, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Direct detect, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Alkaline phosphatase conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Avantor (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Avantor (1 mentions)
Anti il 1β, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved parp 1, by Cell Signaling Technology (1 mentions)
Anti phospho p44 42, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Hrp substrate, by Thermo Fisher Scientific (1 mentions)
Anti mmp2, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit and anti mouse secondary antibodies, by Merck Group (1 mentions)
8 protocols using novex ap chromogenic substrate bcip nbt
1
Western Blot Analysis of TMV
2
Western Blot for Protein Detection
3
STAT3 and STAT6 Activation in PBMCs
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PBMCs were cultured for 24 h with RH, RHΔrop16 or RHΔrop18 strains. Then, cells were lysed in RIPA buffer (Amresco, USA) containing a protease inhibitor cocktail (Amresco) and phosphatase inhibitor (Sigma-Aldrich, Darmstadt, Germany). Equivalent amounts of protein were electrophoresed on 10% SDS polyacrylamide gels and then electroblotted onto 0.45 μm nitrocellulose membranes (10600007, GE healthcare). After blocking with 3% milk protein for 30 min at room temperature, the membranes were incubated with the following primary antibodies: anti-phopho-STAT3 (Tyr 705) (Abcam, UK), anti-phopho-STAT6 (Tyr 641) (Abcam), and anti-IL1β (Santa Cruz Biotechnology, USA). The membranes were then washed three times with PBS containing Tween 20 and incubated with polyclonal goat anti-rabbit IgG conjugated with alkaline phosphatase (Sigma-Aldrich, Darmstadt, Germany) for 1 h at room temperature. Positive reactions were detected using Novex® AP Chromogenic Substrate BCIP/NBT (Thermo Fisher, USA). Densitometry analysis was conducted using ImageJ (Schneider et al., 2012 (link)), and the signal value from each band was normalized using β-actin (Ambion, USA) as the normalization protein. PBMCs from 3 individuals were randomly selected from each group and the normalized protein expression levels were plotted in a histogram.
4
Western Blot Analysis of Protein Expression
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Cells (2 × 106) were seeded on Petri dishes and induced as described previously. After the incubation time, the cells were detached, and the protein was isolated with RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and 1 mM PMSF (Sigma-Aldrich). Protein concentration was determined by DirectDetect® (Merck Millipore). Thirty micrograms of proteins were separated on 12% polyacrylamide gel (120 V) and transferred on PVDF membranes with wet transfer (100 V, 400 mA, 70 min). Membranes were then blocked in 5% fat-free milk in TBST buffer prior to overnight incubation in 4 °C in the primary antibodies, anti-Akt (#9272S), anti-phospho-Akt (#4060S), anti-p44–42 (Erk1/2, #4695S), anti-phospho-p44–42 (#4370S), anti-SOD1 (#71G8), anti-cleaved PARP1 (#5625, Cell Signaling Technology), and anti-GAPDH (1:1000, sc-59,540, SantaCruz Biotechnology), as a reference. After incubation, the membranes were washed three times with TBST buffer and incubated with secondary antibodies (1:15000, Sigma-Aldrich) for 4 hours at 4 °C. The membranes were washed once again, and the bands were visualized by using Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific). Densitometric analysis was conducted with ImageJ. The experiment was conducted in triplicate.
5
Western Blot Analysis of Cellular Proteins
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The cells (1 × 106) were seeded on Petri dishes, incubated to reach 90% confluency and induced as described previously. Protein isolation, electrophoresis, transfer and Western blot procedure were conducted as described previously [12 (link)]. Primary antibodies were used, i.e., anti-Akt (1:1000 in 5% BSA, Cell Signaling), anti-p44/42 (1:1000 in 5% BSA, Cell Signaling, Danvers, MA, USA), anti-SOD-1 (1:200, Cell Signaling, Danvers, MA, USA) or anti-GAPDH (1:1000, SantaCruz Biotechnology, Dallas, TX, USA), as a reference. Bands were visualized with the Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific, Waltham, MA, USA). A densitometric analysis was conducted in ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The experiment was conducted in triplicate.
6
Western Blot Analysis of Protein Expression
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Cells (2 × 106) were seeded on 100-mm Petri dishes. The next day, cells were treated with DON and LY294002 as described above. After 24 h cells were harvested and frozen in − 80 °C. Protein was isolated using RIPA buffer with PMSF, protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The concentration of protein was measured with DirectDetect® (Merck Millipore, Burlington, MA, USA). 30 µg of protein was used for electrophoresis (separation 12% polyacrylamide gel-120 V) and wet transfer (PVDF membranes, 100 V, 400 mA, 70 min). Then, membranes were blocked with 5% fat-free milk in TBST buffer for 1 h, washed three times in TBST for 5 min and then incubated overnight (4 °C) with primary antibodies. After incubation, the membranes were washed in TBST buffer as described above and the secondary (1:15,000) antibodies were added for 4 h (4 °C). Then, membranes were ones again washed in TBST, next, bands were visualized with Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific Inc, Waltham, MA, USA).
7
Purification of Nesprin-2/Telethonin Complex
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The protein elution above was loaded onto a Superdex 200 16/60 column equilibrated with a buffer of 50 mM Tris/HCl pH7.5; 150 mM NaCl; 20 mM βME. The column was run at 2 ml/min flow rate, and fractions of 2 ml were collected, and an aliquot was run on an SDS-PAGE. The peak (fractions 101–112) that contained nesprin-2/telethonin complex were pooled and loaded onto a Superdex 75 10/300 column for further purification. The column was equilibrated with the same buffer, and the βME was increased to 100 mM to reduce aggregation. The column was run at a 0.5 ml/min flow rate, and fractions were collected. The complex elutes as a single peak at 10.29 ml. An aliquot of this elution was run onto 8 to 16% SDS-PAGE and blotted to a PVDF membrane. The membrane was first probed with nesprin-2 N3 antibody and developed with HRP substrate (Thermo Fisher Scientific) and visualized on X-ray film. Then the membrane was stripped and then incubated with telethonin primary antibody (sc-25327, Santa Cruz) and a secondary mouse antibody conjugated with alkaline phosphatase, then visualized with Novex AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific).
8
Protein Expression Analysis by Western Blot
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The cells were seeded on Petri dishes and induced as described previously. After 72 h, the cells were detached and the protein was isolated with RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA) as described previously [12 (link)]. Membranes were blocked in 5% fat-free milk in TBST buffer prior to overnight incubation in 4 °C in the primary antibodies anti- MMP-2 (1:200 in 1% fat-free milk, SantaCruz Biotechnology, Dallas, TX, USA) or anti-GAPDH (1:1000, SantaCruz Biotechnology, Dallas, TX, USA) as a reference. After incubation, the membranes were washed three times with TBST buffer and incubated with anti-rabbit and anti-mouse secondary antibodies (1:15,000, Sigma-Aldrich, Saint Louis, MO, USA) for MMP-2 and GAPDH, respectively, for four hours at 4 °C. The membranes were washed once again and the bands were visualized with using Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific, Inc, Waltham, MA, USA). Densitometric analysis was conducted with ImageJ [36 ] (National Institutes of Health, Bethesda, MD, USA). The experiment was conducted in triplicate.
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