Ubiquitylation assay was carried out using an ubiquitinylation kit (ENZO Life Sciences, Farmingdale, NY). The standard reaction mixture (20 μl) contained the following components: ubiquitinylation buffer, 20 U/ml inorganic pyrophosphate solution, 1 mM DTT, 5 mM Mg-ATP, 100 mM E1, 2.5μM E2 (UBcH5a, -b and -c), 2.5μM biotinylated ubiquitin and 0.2 μM of the target proteins (IAPs). The reactions were incubated at 37°C for 1 h, stopped by adding an equal volume of 2× Laemmli sample buffer and boiling at 95°C, and subjected to SDS-PAGE followed by immunoblot analyses using anti-HA antibody or Streptavidin–Alkaline Phosphatase (AP) conjugate (Roche Applied Science).
Streptavidin alkaline phosphatase conjugate
Manufactured by Roche
Sourced in United Kingdom, Germany, Italy, United States
Streptavidin-alkaline phosphatase conjugate is a laboratory reagent that combines the high-affinity binding properties of streptavidin with the enzymatic activity of alkaline phosphatase. This conjugate is commonly used in various biotechnological and diagnostic applications that involve the detection and quantification of target analytes.
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Lab products found in correlation
Maxisorp nunc immunoplates, by Thermo Fisher Scientific (3 mentions)
3550 uv microplate reader, by Bio-Rad (3 mentions)
Ubiquitinylation kit, by Enzo Life Sciences (1 mentions)
Nbt bcip, by Roche (1 mentions)
Pathscan phospho vegfr 2 tyr1175 sandwich elisa kit, by Cell Signaling Technology (1 mentions)
Ab6720, by Abcam (1 mentions)
Ab181421, by Abcam (1 mentions)
Recombinant human ptn, by R&D Systems (1 mentions)
Ez link sulfo nhs biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin fraction 5, by Roche (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
8 protocols using streptavidin alkaline phosphatase conjugate
1
Ubiquitylation Assay for IAPs
2
Confirmation of RT-qPCR Results Using REBA HPV-ID
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To confirm the results of the RT-qPCR assays, the REBA HPV-ID test (YD Diagnostic, Yongin, Republic of Korea) based on PCR-REBA was performed according to the manufacturer's instructions. 14 For the REBA HPV-ID, hybridization and washing processes were performed according to the manufacturer's instructions. In brief, the biotinylated PCR products were denatured at 25 8C for 5 min in denaturation solution and diluted in 970 ml of hybridization solution on the REBA membrane strip in the blotting tray provided. Denatured single-stranded PCR products were hybridized to the probes on a strip at 50 8C for 30 min. The strips were then washed twice with gentle shaking in 1 ml of washing solution for 10 min at 50 8C, incubated at 25 8C with 1:2000 diluted streptavidin-alkaline phosphatase (AP) conjugate (Roche Diagnostics, Mannheim, Germany) in conjugate diluents solution (CDS) for 30 min, and finally washed twice with 1 ml CDS at room temperature for 1 min. The colorimetric hybridization signals were visualized by the addition of a 1:50 dilution of AP-mediated staining solution, NBT/BCIP (Roche Diagnostics), and incubation until a color change was detected. Finally, the band pattern was read and interpreted.
3
Quantifying VEGF-A Secretion and VEGFR-2 Phosphorylation
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Semi-confluent tumor cell cultures were incubated in 0.1% BSA/RPMI-1640 medium without FBS for 24 h. Culture supernatants were collected, centrifuged at 600× g for 10 min to remove cells in suspension, concentrated at least ten-fold in Centriplus concentrators (Amicon, Beverly, MA, USA) and frozen at −20 °C till use. Cells were detached from the flasks with a solution of 1 mM EDTA in PBS and the total cell number/culture was recorded. Quantification of the amount of VEGF-A in the concentrated supernatants was performed using Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark) coated with goat anti-VEGF-A IgGs, as previously described [31 (link)]. Briefly, detection of the cytokines was performed with biotinylated goat anti-VEGF (R & D Systems, Abingdon, UK) and streptavidin-alkaline phosphatase conjugate (1:10,000) (Roche). The reaction was stopped and optical density at 405 nm was measured in a Microplate reader 3550-UV (Bio-Rad, Hercules, CA, USA).
Modulation of VEGFR-2 phosphorylation in response to VEGF-A in untreated cells or cells exposed to EA was analyzed using the PathScan® Phospho-VEGFR-2 (Tyr1175) Sandwich Elisa Kit (Cell Signaling Technology, Danvers, MA, USA).
Modulation of VEGFR-2 phosphorylation in response to VEGF-A in untreated cells or cells exposed to EA was analyzed using the PathScan® Phospho-VEGFR-2 (Tyr1175) Sandwich Elisa Kit (Cell Signaling Technology, Danvers, MA, USA).
4
Serum PTN and TNF-α Quantification
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The blood samples were collected via venipuncture into tubes containing anticoagulants. The protein level of PTN in serum was determined on covered 96-well ELISA plates, as previously reported (32 (link)). Rabbit anti-human PTN monoclonal antibodies (1:2,000, ab14025; Abcam, Cambridge, MA, USA) were diluted in Tris-buffered saline (TBS) and incubated at 4°C overnight. Biotinylated affinity-purified anti-rabbit secondary antibody (1:50,000, ab6720; Abcam) was added into the wells and incubated at room temperature for l h. Streptavidin/alkaline phosphatase conjugate (Roche Diagnostics GmbH, Mannheim, Germany) was added and maintained at room temperature. The absorbance at 405 nm was measured on a plate reader. Recombinant human PTN (R&D Systems, Inc.) was used as a standard control. The concentration of TNF-α was also measured using an ELISA kit (ab181421; Abcam) according to the protocols. The kit mainly consists of anti-TNF-α antibodies and the regent for the ELISA assay. The absorbance of TNF-α was determined at 450 nm.
5
Quantification of VEGF-A and PlGF in GBM Cell Secretome
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Conditioned media from GBM cells were obtained by incubating semi-confluent cultures for 24 h in 0.1% BSA/DMEM medium without FBS. These conditions did not significantly affect cell viability. Supernatants were concentrated at least 10-fold in Centriplus concentrators (Amicon, Beverly, MA). Cells were detached from the flasks with PBS/EDTA. Cytokine secretion values were normalized by the total number of cells.
Quantification of the amount of VEGF-A and PlGF in the conditioned medium was performed using goat anti-VEGF-A or anti-PlGF IgGs (R&D Systems, Abingdon, UK), at a concentration of 10 μg/ml in PBS, to coat Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark). Detection of the cytokines was performed with biotinylated goat anti-VEGF or anti-PlGF IgGs (0.4 μg/ml; R&D Systems) followed by incubation with streptavidin alkaline phosphatase conjugate (1:10,000) (Roche, Monza, Italy) and alkaline phosphatase reaction. Optical density at 405 nm was measured in a 3550-UV Microplate reader (Bio-Rad, Hercules, CA).
Quantification of the amount of VEGF-A and PlGF in the conditioned medium was performed using goat anti-VEGF-A or anti-PlGF IgGs (R&D Systems, Abingdon, UK), at a concentration of 10 μg/ml in PBS, to coat Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark). Detection of the cytokines was performed with biotinylated goat anti-VEGF or anti-PlGF IgGs (0.4 μg/ml; R&D Systems) followed by incubation with streptavidin alkaline phosphatase conjugate (1:10,000) (Roche, Monza, Italy) and alkaline phosphatase reaction. Optical density at 405 nm was measured in a 3550-UV Microplate reader (Bio-Rad, Hercules, CA).
6
Binding Assay for SubB Glycoprotein
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Purified SubBΔS106/ΔT107 was labelled with biotin using the EZ-Link® Sulfo-NHS-Biotinylation Kit (Thermo Scientific) according to the manufacturer’s instructions. Purified human and bovine α−1 acid glycoprotein (Sigma cat. nos G9885 and G3643) were dissolved in water at 5 mg/ml and 5 μl volumes of serial two-fold dilutions were spotted onto nitrocellulose filters and air dried at 37 °C overnight. Filters were then blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 (TTBS) for 2 h. After washing three times in TTBS, filters were overlaid with 1 μg/ml biotin-SubBΔS106/ΔT107 in TTBS and incubated overnight at 4 °C. Filters were then washed three times in TTBS and bound biotin-SubBΔS106/ΔT107 was detected using streptavidin-alkaline phosphatase conjugate (Roche). Filters were developed using a chromogenic nitro-blue tetrazolium/X-phosphate substrate system (Roche).
7
Quantification of VEGF-A and PlGF Binding to VEGFR-1
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Quantification of VEGF-A and PlGF binding to VEGFR-1 was performed on 96-well Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark) coated with 10 μg/ml VEGFR-1/Fc chimera in PBS. After blocking of the plates with 3% BSA (w/v) in PBS (hereafter referred to as blocking buffer), 50 μl of PlGF or VEGF-A solution (50 ng/ml in blocking buffer) were added to selected wells and incubated for 2 h. Detection of the amount of VEGFR-1/Fc chimera bound cytokine was performed with biotinylated goat anti-VEGF-A or anti-PlGF antibodies and streptavidin-alkaline phosphatase-conjugate (1:10,000; Roche). After alkaline phosphatase reaction, optical density at 405 nm was measured in a Microplate reader 3550-UV (Bio-Rad, Hercules, CA). The specificity of VEGF-A or PlGF binding to VEGFR-1 was demonstrated by preincubating the growth factors with neutralizing antibodies, before adding them to the wells coated with VEGFR-1/Fc chimera, and background was determined in blocking buffer coated wells.
The effect of D16F7 mAb on VEGF-A or PlGF binding to VEGFR-1 was evaluated by pre-incubating selected sVEGFR-1 coated wells with 10 μg/ml of D16F7 or control mAb for 30 min at room temperature, before adding the growth factors to the plate.
The effect of D16F7 mAb on VEGF-A or PlGF binding to VEGFR-1 was evaluated by pre-incubating selected sVEGFR-1 coated wells with 10 μg/ml of D16F7 or control mAb for 30 min at room temperature, before adding the growth factors to the plate.
8
Quantitative IgE ELISA Assay Protocol
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Plasma/serum total IgE concentrations were measured in March 2014 by ELISA. Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight with shaking with 1000× diluted polyclonal rabbit anti-human IgE capture antibody (Dako, Glostrup, Denmark) in 0.1 M sodium bicarbonate buffer, pH 9.6, at 4 °C, and blocked with shaking for 1 h with 2% bovine serum albumin fraction V (Roche, Mannheim, Germany)/phosphate buffered saline at room temperature. Plates were then incubated with shaking for 1 h with 100 μL of 20× and 200× diluted plasma/serum samples and control samples at room temperature. The WHO 3rd international standard of human serum IgE (NIBSC) was applied at a 10-step 2× serial dilution starting at 200 ng/mL. After washing, the plates were incubated with shaking for 1 h with 1000× diluted biotinylated goat anti-human IgE, ε-chain specific, detection antibody (Vector Laboratories, Burlingame, CA, USA) followed by 3000× diluted streptavidin-alkaline phosphatase conjugate (Roche) for 1 h at room temperature, and by 100 μL 1 mg/mL p-nitrophenylphosphate substrate (Roche) in 0.1 M diethylanolamine buffer for 25 min. The reaction was stopped by 100 μL 3 M NaOH and absorbance was measured at 405 nm. The detection limit was 4.0 ng/mL.
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