Western blots were performed on ~10 μg BSA or 60 μg BSA-PA. Due to the diffuse banding pattern of BSA-PA, increased amounts of BSA-PA (60 μg) were loaded for the western blot (Figure 2(c) ) in order to detect differences between samples. The proteins were separated on a 12% acrylamide gel at 120 V and then transferred to PVDF membranes and blocked with 5% nonfat milk solution in Dulbecco's PBS (Caisson Labs, Logan, UT) for 1 hr at room temperature. After blocking, the membrane was incubated in primary antibody (complete patient serum) diluted 1 : 100 in 5% nonfat milk solution overnight at 4°C. The membrane was washed three times with PBS + 0.02% Tween 20 and incubated with anti-human IgG conjugated to alkaline phosphatase (Sigma) diluted 1 : 5,000 for 1 hr at room temperature. The membrane was washed three times with PBS + 0.02% Tween 20 and then one time with PBS and the protein bands were visualized on photographic film (Kodak) using Novex AP Chemiluminescent Substrate (Invitrogen).
Novex ap chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Novex AP Chemiluminescent Substrate is a laboratory reagent used for detecting and quantifying alkaline phosphatase (AP) in various applications, including Western blotting, ELISA, and other immunoassays. The substrate emits light upon reaction with the AP enzyme, allowing for sensitive and quantitative detection of target proteins or analytes.
Automatically generated - may contain errors
Lab products found in correlation
Photographic film, by Kodak (1 mentions)
Anti human igg conjugated to alkaline phosphatase, by Merck Group (1 mentions)
Fluorcheme imager, by Cell Biosciences (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab88770, by Abcam (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
P vegfr2, by Cell Signaling Technology (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Tvegfr2, by Cell Signaling Technology (1 mentions)
P plcγ1, by Cell Signaling Technology (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
4 protocols using novex ap chemiluminescent substrate
1
Western Blot Visualization of BSA-PA
2
Immunoblotting Procedure for Hippocampal Protein Analysis
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Immunoblotting has been described previously [10 (link),11 (link)]. A bicinchoninic acid (BCA; Thermo Fisher Scientific, Waltham, MA, USA) protein assay using bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) as a standard was used to determine protein concentrations. Hippocampal protein samples were heated for 5 min to 95 °C (GLT-1, actin, CP13, MC1, and PHF1) or not heated (vGLUT1, PSD95, Tau-5, HT7, synaptophysin, actin, GSK3β, pGSK3β) and loaded on 10% hand-cast sodium dodecyl sulfate (SDS)-page gels. After transfer, membranes were blocked for 1 h at room temperature (∼23 °C) and then incubated with a primary antibody (see Table 1 for list of antibodies) directed against the protein of interest overnight at 4 °C. The next day, membranes were incubated with the appropriate biotinylated or horseradish peroxidase conjugated secondary antibody for 1 h at room temperature (∼23 °C). Blots were then incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) or Novex AP chemiluminescent substrate (Invitrogen) for 5 min and visualized using Fluorchem E imager (Cell Biosciences, Preston VIC, Australia).
3
Bovine Embryo Western Blot Analysis
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Bovine in vitro-produced embryos at the 1-cell (n=400) and blastocyst (n=10) stages were lysed in NuPAGE LDS
Sample Buffer with Reducing Agent (Invitrogen) in a 20 µl volume and boiled for 5 min. The samples were subjected to SDS-PAGE
through a 4–12% Bis-Tris gel and transferred to a PVDF membrane using an iBlot Dry Blotting System (Invitrogen). The blotted
membrane corresponding to 30–60 kDa was blocked with 10% (v/v) FCS in PBS containing 0.05% (v/v) Tween 20 (PBST) for 30 min. A
rabbit polyclonal antibody to CTR (ab103422, Abcam, Cambridge, UK) was diluted 200 times with PBST containing 5% (v/v) FCS and
mounted onto the membrane for 1 h. After extensive washing with PBST, the membrane was treated for 30 min with 2,000 times-diluted
alkaline phosphatase-conjugated bovine anti-rabbit IgG (sc-2372, Santa Cruz Biotechnology, Dallas, TX, USA). After washing, the
signal was developed for 5 min with Novex AP Chemiluminescent Substrate (Invitrogen) and exposed to FP-3000B film (Fujifilm,
Tokyo, Japan). The same procedure except for the use of an anti-Histone H2A antibody (1,000-times diluted, ab88770, Abcam) was
applied to the membrane corresponding to 10-30 kDa to assess the expression levels of Histone H2A as a loading control.
Sample Buffer with Reducing Agent (Invitrogen) in a 20 µl volume and boiled for 5 min. The samples were subjected to SDS-PAGE
through a 4–12% Bis-Tris gel and transferred to a PVDF membrane using an iBlot Dry Blotting System (Invitrogen). The blotted
membrane corresponding to 30–60 kDa was blocked with 10% (v/v) FCS in PBS containing 0.05% (v/v) Tween 20 (PBST) for 30 min. A
rabbit polyclonal antibody to CTR (ab103422, Abcam, Cambridge, UK) was diluted 200 times with PBST containing 5% (v/v) FCS and
mounted onto the membrane for 1 h. After extensive washing with PBST, the membrane was treated for 30 min with 2,000 times-diluted
alkaline phosphatase-conjugated bovine anti-rabbit IgG (sc-2372, Santa Cruz Biotechnology, Dallas, TX, USA). After washing, the
signal was developed for 5 min with Novex AP Chemiluminescent Substrate (Invitrogen) and exposed to FP-3000B film (Fujifilm,
Tokyo, Japan). The same procedure except for the use of an anti-Histone H2A antibody (1,000-times diluted, ab88770, Abcam) was
applied to the membrane corresponding to 10-30 kDa to assess the expression levels of Histone H2A as a loading control.
4
VEGFR-2 Signaling Pathway Analysis
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Serum starved PAE-KDR cells were incubated for 10 min with 1.5 nM VEGF (in all experiments VEGF-A165 was used) with or without 30 min pretreatment with scFvs. Cells were lysed with lysis buffer (50 mM Tris (pH 7.5), 100 mM NaCl, 0.5% (w/v) Triton X-100) supplemented with protease inhibitor cocktail (Complete Mini EDTA-free, Roche) and phosphatase inhibitors (200 μM Na3VO4, 20 μM phenylarsine oxide). Lysates were diluted with 5× loading buffer (0.25 M Tris-HCl pH 6.8, 0.5 M DTT, 10% SDS, 50% glycerol, 0.5% bromophenol blue). Samples were boiled at 50 °C for 30 min, and resolved by 7% SDS PAGE, transferred to PVDF membranes (GE Healthcare, Piscataway, NJ, USA), and immunodecorated with primary antibodies (dilution 1:1000), followed by secondary alkaline phosphatase-coupled antibodies (1:10000). Immunoblots were developed with Novex AP Chemiluminescent Substrate (Invitrogen, Carlsbad, CA, USA). Amersham Imager 600 (GE) was used for analysis. Antibodies used were as follows: pVEGFR-2 (2478, Cell Signaling), tVEGFR-2 (2479, Cell Signaling), pPLCγ1 (2821, Cell Signaling), tPLCγ1 (2822, Cell Signaling), pAKT (4060, Cell Signaling), tAKT (4051, Cell Signaling), p38 (4631, Cell Signaling), and tp38 (9212, Cell Signaling). Protein marker used was PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (26619, ThermoFisher, Waltham, MA, USA).
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