The following antibodies (Abs) and other materials were used: phycoerythrin (PE)-conjugated anti-human HLA-A, B, C (W6/32, BioLegend), PE-conjugated anti-human MICA (159227, R&D Systems), PE-conjugated anti-CD107a (H4A3, SouthernBiotech), PE-conjugated anti-human IgG (polyclonal, Jackson ImmunoResearch), FITC-conjugated anti-KIR2DL2 (CH-L, BD Biosciences), FITC-conjugated anti-human IgG (polyclonal, Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), allophycocyanin (APC)-conjugated anti-mouse IgG (polyclonal, Jackson ImmunoResearch), Pacific Blue-conjugated anti-human CD16, purified/biotinylated anti-SUDV-GP (3C10), purified anti-B7H6, Capture: purified anti-human IFN-γ (NIB42, BioLegend), Detection: biotin anti-human IFN-γ (4S.B3, BioLegend), 7-aminoactinomycin D (7AAD) (BioLegend), anti-phosphotyrosine 4G10, anti-PLCγ1 (Upstate), anti-SHP-1 (Santa Cruz), anti-GAPDH (Biodesign), and p-nitrophenyl phosphate (pNPP; New England BioLabs). The following fusion-Igs were used as previously described; NKG2D-Ig (37 (link)), NKp30-Ig (38 (link)), NKp44-Ig (39 (link)), NKp46-Ig (40 (link)), KIR2DL4-Ig (41 (link)), KIR2DL1-Ig (27 (link)), and KIR2DL2-Ig (42 (link)).
P nitrophenyl phosphate pnpp
Manufactured by New England Biolabs
Sourced in United States
P-nitrophenyl phosphate (pNPP) is a colorless, water-soluble compound that is commonly used as a substrate for various enzymatic assays. It serves as a chromogenic substrate for the detection and quantification of phosphatase enzyme activity.
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Lab products found in correlation
7 aminoactinomycin d 7 aad, by BioLegend (1 mentions)
Alexa fluor 647 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Anti shp1, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated anti human igg, by Jackson ImmunoResearch (1 mentions)
Pe conjugated anti human igg, by Jackson ImmunoResearch (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Okadaic acid, by Santa Cruz Biotechnology (1 mentions)
5 protocols using p nitrophenyl phosphate pnpp
1
Characterization of NK Cell Receptor Interactions
2
Colorimetric Assay for Alkaline Phosphatase Activity
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The purified His(6)-tag fused Aa-mALP protein was assayed for its phosphatase activity using p-nitrophenyl phosphate (pNPP) (New England BioLabs, Ipswich, MA, USA) as a colorimetric substrate. The hydrolysis of 5 mM pNPP to p-nitrophenol (pNP), a water-soluble yellow product with strong absorbance at 405 nm in ALP buffer (5 mM MgCl2, 100 mM NaCl, and 100 mM Tris-HCl, pH 9.5) was assessed at 25 °C, using a microplate spectrophotometer. The specific activity of ALP was expressed as the release of pNP in µmole/min/mg of protein.
3
Colorimetric Assay for Alkaline Phosphatase
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A colorimetric assay using the substrate p-Nitrophenyl phosphate (PNPP) (New England Biolabs) was used to quantify alkaline phosphatase activity in colonies grown on MOPS agar plates with no added BCIP using the inoculation regime described above. After 12–24 h incubation, the colonies were scraped from the agar with a pipette tip and resuspended in 1 mL 10 mM Tris-HCL buffer at pH 8. Fifty μL of the cell suspension was mixed with 50 μL of room temperature 1-step PNPP solution or 50 μL of Tris-HCL buffer at pH 8 and incubated for 1 h. A405 and A600 were recorded at t = 0 min and t = 60 min. AP activity was calculated using the equation 1000 × (ΔA405/(time (min) × A600)) where ΔA405 = (A405PNPP – A405NoPNPP)t60 – (A405PNPP – A405NoPNPP)t0.
4
Recombinant L. major PP5 Phosphatase Assay
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Activity of L. major recombinant PP5 was measured in a phosphatase assay using p-nitrophenyl phosphate (pNPP) (New England Biolabs) as a substrate. Amounts of recombinant protein were quantified using a Bradford assay. As GST alone can alter protein quantification, samples were further compared with known BSA concentrations using densitometry analysis of a SimplyBlue SafeStain (Invitrogen) stained gel. 1 ug of each recombinant sample, diluted in 1× colorimetric assay buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.02% β-mercaptoethanol and 0.1 mg/mL bovine serum albumin [BSA]), was added to corresponding wells on a 96-well plate and the reaction was initiated by addition of pNPP. After a 45-min incubation at room temperature, 5N NaOH was added to each well to stop the reaction. Absorbance (ABS) was read using a plate reader at 405 nm. Okadaic acid (OA) and arachidonic acid (AA) were purchased from Santa Cruz and Sigma, respectively. Statistical analysis was performed with GraphPad Prism v. 6.04.
5
SARS-CoV-2 Nucleocapsid Protein Detection
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The first antibody (i.e., capture side) and the second antibody (i.e., detection side) for SARS-CoV-2 nucleocapsid proteins were purchased from Meridian Life Science, Memphis, TN, U.S.A. (Cat. Nos. 9548 and 9547, respectively). The recombinant antigen for SARS-CoV-2 nucleocapsid proteins was obtained from EastCoast Bio, North Berwick, ME, U.S.A. (Cat. No. LA600). ALP and NADH were purchased from Roche (Basel, Switzerland). 3α-HSD was purchased from Asahi Kasei Pharma (Tokyo, Japan). The thio-NAD was obtained from Oriental East (Tokyo, Japan). 17β-Methoxy-5β-androstan-3α-ol 3-phosphate was synthesized by one of the authors (T.Y.). p-Nitrophenylphosphate (p-NPP) was purchased from New England Biolabs (Ipswich, MA, U.S.A.). SARS-CoV-2 (JPN/TY/WK-521) was propagated in Vero-E6/TMPRSS2 (JCRB1819) cells using Dulbecco's modified Eagle's medium (DMEM) solution with 2% fetal calf serum, whose viral infectivity was 5.25 log 10 tissue culture infectious dose 50 (TCID 50 )/50 µL and RNA amount was 2.6 × 10 7 copies/µL. The culture supernatant (hereafter referred to as virions) was inactivated by UVB irradiation at 51 µW/cm 2 for 30 min (GL15, Toshiba Lighting & Technology, Yokosuka, Japan). The other chemicals and the disposable plasticware were of commercial grade.
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