Substrate reagent

Manufactured by R&D Systems

Sourced in United States

Substrate reagent is a laboratory product designed to detect and measure the presence or activity of specific enzymes or substrates. It provides a reagent solution that changes color or generates a measurable signal in the presence of the target analyte. The core function of the substrate reagent is to facilitate quantitative or qualitative analysis of the substance of interest.

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Lab products found in correlation

Ezq protein quantitation kit, by Thermo Fisher Scientific (2 mentions) Anti sdf 1 capture antibody, by R&D Systems (2 mentions) Maxisorp 96 well plate, by Thermo Fisher Scientific (2 mentions) Streptavidin coated 96 well plate, by Thermo Fisher Scientific (2 mentions) Microplate reader, by Bio-Rad (2 mentions) Mouse il 17 duoset elisa kit, by R&D Systems (2 mentions) Abts elisa development kit, by Thermo Fisher Scientific (2 mentions) Streptavidin hrp, by Jackson ImmunoResearch (1 mentions) High binding half area 96 well plates, by Corning (1 mentions) Recombinant murine tnf, by Thermo Fisher Scientific (1 mentions) Streptavidin horseradish peroxidase, by R&D Systems (1 mentions) Complete lysis m edta free buffer, by Roche (1 mentions) Anti sdf 1α, by R&D Systems (1 mentions) Streptavidin conjugated horseradish peroxidase, by R&D Systems (1 mentions) Biotin labeling kit nh2, by Dojindo Laboratories (1 mentions) Thermo multiskan ex, by Thermo Fisher Scientific (1 mentions) Elisa plate, by Thermo Fisher Scientific (1 mentions) Donkey anti mouse igg hrp, by Jackson ImmunoResearch (1 mentions) Streptavidin horseradish peroxidase hrp, by R&D Systems (1 mentions) Peroxidase conjugated avidin, by Agilent Technologies (1 mentions)

Spelling variants of this product

Substrate Reagent Pack (19 citations) Substrate Reagent Pack DY999 (7 citations) Substrate reagent solution (6 citations) TMB substrate reagent (2 citations) TMB substrate Reagent Pack (2 citations)

15 protocols using substrate reagent

Measuring Neutrophil Infiltration in Eyes

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The number of neutrophils in the eye was estimated by measuring MPO activity as described previously, with slight modification. Briefly, eyes isolated 24 h post-infection were homogenized in 1.0 mL phosphate-buffered saline, the homogenate was centrifuged (10,000× g, 15 min, 4 °C), the resulting pellet was suspended in 0.03 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 50 mM hexadecyltrimethylammonium bromide, and 0.07 mL of 50 mM potassium phosphate (pH 6.0) was then added to the suspension. Each sample was subjected to three freeze–thaw cycles and then centrifuged at 10,000× g for 10 min at 4 °C, after which 0.02 mL of the resulting supernatant was added to 0.05 mL of substrate reagent (R&D Systems, Minneapolis, MN, USA), and the mixture was incubated for 20 min at room temperature. The reaction was terminated by adding 25 µL of 2 N H₂SO₄, and the absorbance at 450 nm was measured.

Kishimoto T., Ishida W., Nasukawa T., Ujihara T., Nakajima I., Suzuki T., Uchiyama J., Todokoro D., Daibata M., Fukushima A., Matsuzaki S, & Fukuda K. (2021). In Vitro and In Vivo Evaluation of Three Newly Isolated Bacteriophage Candidates, phiEF7H, phiEF14H1, phiEF19G, for Treatment of Enterococcus faecalis Endophthalmitis. Microorganisms, 9(2), 212.

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TNF-alpha Quantification by ELISA

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High-binding half area 96-well plates (Corning) were coated with 25uL of unconjugated anti-TNF antibody (Invitrogen; clone 1F3F3D4) at a concentration of 2ug/ml in PBS and incubated overnight at 4⁰C. Plates were then washed 6 times with PBST (1X PBS + 0.05% Tween-20) and blocked for 1hr at room temperature with 120uL of 1X PBS + 5% FCS. Next, the blocking solution was removed and 25uL of indicated macrophages supernatants plus a standard curve using recombinant murine TNF (Peprotech) in 1xPBS + 5% FCS were added to the plates and incubated for an hour at room temperature. Plates were washed as above, and then 25uL of biotinylated anti-TNF (Invitrogen; clone MP6-XT3) was added a concentration of 1ug/ml in PBS + 5% FCS for 1hr at room temperature. Plates were then washed, and 25uL of streptavidin-HRP (Jackson ImmunoResearch) was added at a concentration of 2.5ug/ml in 1xPBS + 5% FCS for 1hr at room temperature. Plates were then washed, assay was developed using a 50uL of Substrate reagent (R&D Systems), and absorbance was read at 450nm.

Dang E.V., Lei S., Radkov A., Volk R.F., Zaro B.W, & Madhani H.D. (2022). Secreted fungal virulence effector triggers allergic inflammation via TLR4. Nature, 608(7921), 161-167.

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SDF-1 Splice Variant-Specific ELISA

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SDF-1 splice variant-specific ELISAs (R&D Systems, Minneapolis, MN) were performed as previously described.^{8 ,21 (link)} Briefly, Tet-Off BMSCs were plated and treated as above. After 24 h, the media were collected and cell lysates prepared in Complete Lysis-M EDTA-free buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN). The anti-SDF-1 capture antibody (R&D Systems) in sodium bicarbonate buffer pH 9.4 was bound to MaxiSorp^™ 96-well plates (Nunc, Thermo Fisher Scientific) overnight. Plates were blocked for 2 h with 1% BSA in PBS. Murine SDF-1α or SDF-1β (PeproTech, Rocky Hill, NJ) standards and samples (1:2 diluted) were incubated for 2 h prior to incubating with the biotinylated anti-SDF-1α and anti-SDF-1β detection antibody (2 h; R&D Systems), respectively. Streptavidin-horseradish peroxidase (R&D Systems) was incubated for 20 min followed by the substrate reagent (R&D Systems) for 20 min. Sulfuric acid (2 N) was added to stop the enzymatic color reaction and absorbance was read at 450 nm. SDF-1α and SDF-1β protein expression was calculated using standard curves and normalized to total protein, which was quantified using the EZQ^® Protein Quantitation Kit (Invitrogen).

Herberg S., Kondrikova G., Hussein K.A., Johnson M.H., Elsalanty M.E., Shi X., Hamrick M.W., Isales C.M, & Hill W.D. (2014). Mesenchymal Stem Cell Expression of Stromal Cell-Derived Factor-1β Augments Bone Formation in a Model of Local Regenerative Therapy. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 33(2), 174-184.

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Quantitative ELISA for Phospho-CSE1L

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Anti-CSE1L (H2) antibody-coated 96-well plates (Costar) were blocked with 5% BSA in TBS (Tris-buffered saline) for 1 h. The wells were washed with TBST (0.05% Tween 20 in TBS) and then incubated with serum samples (sixfold dilution with TBS) for 1 h. After being washed with TBST, the wells were incubated with biotin-conjugated anti-phospho-CSE1L antibodies for 1 h. The biotin-conjugated anti-phospho-CSE1L antibodies were prepared by biotinylating anti-phospho-CSE1L antibodies using the Biotin Labeling Kit-NH2 (Dojindo Laboratories, Kumamoto, Japan). The wells were then washed with TBST, reacted with streptavidin-conjugated horseradish peroxidase (R&D Systems, Minneapolis, MN, USA), and followed by incubation with the substrate reagent (R&D Systems). For calibration, two blank wells containing TBST were used as the background value, and two wells that were not coated with anti-phospho-CSE1L antibodies but reacted with all other ELISA reagents were used as control wells. The absorbance at 450 nm was measured within 20 min following the reaction by using a Thermo Multiskan EX microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was assayed two times.

Lee W.R., Shen S.C., Shih Y.H., Chou C.L., Tseng J.T., Chin S.Y., Liu K.H., Chen Y.C, & Jiang M.C. (2015). Early decline in serum phospho-CSE1L levels in vemurafenib/sunitinib-treated melanoma and sorafenib/lapatinib-treated colorectal tumor xenografts. Journal of Translational Medicine, 13, 191.

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Plasma C3 Activation ELISA Assay

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Plasma samples were harvested from mice using sterile technique. ELISA
plates (cat# 3855, ThermoFisher Scientific, Waltham, MA) were coated
with LPS (2 µg/well, catalog# L2762 Sigma-Aldrich, St. Louis,
MO) at 4°C overnight. After washing 3× in buffer
(PBS/0.05% Tween 20) and blocking with BSA (250 µl/well,
1% in PBS buffer), diluted mouse plasma (1:5 or 1:10 in
Mg²⁺-EGTA buffer) was incubated on an ELISA plate at 37°C for
1 h (50 µl/well). The plates were washed 3× in washing buffer
and then goat anti-mouse C3 Ab (100 µl/well, 1:4000 dilution in
1% BSA in PBS buffer; MP Cappel, CA) was added for 1 h. Wells were
washed 3× as above and incubated with HRP-conjugated anti-goat IgG (100
µl/well, 1:2000 dilution in 1% BSA in PBS buffer; Jackson
ImmunoResearch, PA) for 1 h. After 3 washes, Substrate Reagent (R&D
Systems, MN) was added for 10 min (100 µl/well). The reaction was
stopped with 50 µl 1 M H₂SO₄, and the OD of
samples was measured at 450 nm. Similar experiments were performed with normal
human serum and FD-depleted human serum supplemented with purified human FD
(CompTech, Tyler, Texas). The primary and second antibodies used in the LPS
assay with human serum are: Mouse anti-human C3d (ThermoScientific, #HYB
005-01-02) and HRP Donkey anti-Mouse IgG (Jackson Immunoresearch)
respectively.

Wu X., Hutson I., Akk A.M., Mascharak S., Pham C.T., Hourcade D.E., Brown R., Atkinson J.P, & Harris C.A. (2018). Contribution of Adipose-derived Factor D/Adipsin to Alternative Pathway Complement Activation: Lessons from Lipodystrophy. Journal of immunology (Baltimore, Md. : 1950), 200(8), 2786-2797.

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Lipigenine™ Stimulates HBD-3 Production

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Example 6

Lipigenine™ was tested for its ability to stimulate an increase in HBD-3 concentration. The HBD-3 standard ABTS ELISA development kit was obtained from PeproTech (Cat #900-K210). ELISA were performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H₂SO₄in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve.

The addition of Lipigenine™ showed increased HBD-3 concentration at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-3 concentration of 29% was observed for a 0.1% Lipigenine™ formulation while an increase in HBD-3 concentration of 18% was observed for a 1% Lipigenine™ formulation. These results are shown in FIG. 6.

US11633451B2. Antimicrobial peptide stimulating cleansing composition (2023-04-25). GOJO Industries, Inc. [US]. Inventors: Kegui Tian [US], Jessica Rae Tittl [US], Venkatesan V. Padyachi [US].

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Quantification of Murine SDF-1α Levels

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SDF-1α ELISAs (R&D Systems) was performed with either BM interstitial fluid or in medium conditioned by BMSCs cell culture as described previously (Herberg et al., 2013 (link)). The anti-SDF-1 capture antibody (R&D Systems) in sodium bicarbonate buffer pH 9.4 was bound to MaxiSorp™ 96-well plates (Nunc, Thermo Fisher Scientific) overnight. Plates were blocked for 2 h with 1% BSA in PBS. Murine SDF-1α (PeproTech, Rocky Hill, NJ, USA) standards and samples (1:2 diluted) were incubated for 2 h prior to incubating with the biotinylated anti-SDF-1α (2 h; R&D Systems). Streptavidin-horseradish peroxidase (HRP) (R&D Systems) was incubated for 20 min followed by the substrate reagent (R&D Systems) for 20 min. Sulfuric acid (2 N) was added to stop the enzymatic color reaction and absorbance was read at 450 nm. SDF-1α protein expression was calculated using standard curves and normalized to total protein, which was quantified using the EZQ® Protein Quantitation Kit (Invitrogen).

Periyasamy-Thandavan S., Herberg S., Arounleut P., Upadhyay S., Dukes A., Davis C., Johnson M., McGee-Lawrence M., Hamrick M.W., Isales C.M, & Hill W.D. (2015). Caloric Restriction and the Adipokine Leptin alter the SDF-1 signaling axis in Bone Marrow and in Bone Marrow Derived Mesenchymal Stem Cells. Molecular and cellular endocrinology, 410, 64-72.

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Lipigenine Stimulates HBD-2 Expression

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Example 5

Lipigenine™ was tested for its ability to stimulate an increase in HBD-2 concentration. The HBD-2 standard ABTS ELISA development kits were obtained from PeproTech (Cat #900-K172). ELISA was performed according to the manufactory instructions of each kit by adding 100 μl/well of culture medium after overnight treatment. The substrate of ELISA reaction was using the substrate reagent from R&D Systems (DY999), and the reactions were stopped by adding 50 μl of 1N H₂SO₄in each well. The Lipigenine™ culture was compared to the control medium which contained no other ingredients. The results were measured using a colorimeter, absorbance was measured at 450 nanometers (nm) within 30 minutes. Wavelength correction was set to 570 nm. The concentration of each sample was calculated using ELISA standard curve.

The addition of Lipigenine™ showed increased HBD-2 concentrations at both 0.1% and 1% Lipigenine™ in solution as compared to the control. An increase in HBD-2 concentration of 7% was observed for a 0.1% Lipigenine™ formulation while an increase in HBD-2 expression of 23% was observed for a 1% Lipigenine™ formulation. These results are shown in FIG. 5.

US11633451B2. Antimicrobial peptide stimulating cleansing composition (2023-04-25). GOJO Industries, Inc. [US]. Inventors: Kegui Tian [US], Jessica Rae Tittl [US], Venkatesan V. Padyachi [US].

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Salp20 Modulation of Complement Activation

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C57BL/6J mice (The Jackson Laboartory) were administered i.p. with various concentrations of Salp20. Plasma samples were collected at various time points (2 to 24 h) for an LPS-dependent AP activation assay [32 (link)–34 (link)]. Briefly, plasma was diluted (1:10 in Mg²⁺-EGTA GVB²⁺ buffer), applied to LPS-coated ELISA plates (2µg /well), and incubated at 37°C for 1 h. After washing, goat anti-mouse C3 Ab (1:4000 dilution, MP Cappel, cat#55463) was added followed by HRP-conjugated anti-goat IgG (1:2000 dilution; Jackson ImmunoResearch), after which substrate reagent (cat#DY999, R&D Systems) was added for 10 min. The reaction was stopped with 1M H2SO4 and the OD of samples measured at 450 nm.
To assess the AP activity in the OVA-induced asthma model, mice were sacrificed on day 17 (see below) and their lungs lavaged 3 times with 1 mL of sterile PBS. Samples were cleared by centrifugation at 14,000 rpm for 20 min at 4°C. Cleared samples were diluted at 1:2 in Mg²⁺-EGTA GVB²⁺ buffer prior to being applied to LPS-coated plates. Post lavage, lungs were harvested and homogenized in 2 mL of cold PBS. Homogenates were cleared twice with centrifugation at 14,000 rpm × 20 min at 4°C. Samples were normalized by protein concentration based on absorbance at 280 nm and diluted 1:2 in Mg²⁺-EGTA GVB²⁺ buffer prior to being applied to LPS-coated plates. AP activity was measured as above.

Hourcade D.E., Akk A.M., Mitchell L.M., Zhou H.F., Hauhart R, & Pham C.T. (2015). Anti-complement activity of the Ixodes scapularis salivary protein Salp20. Molecular immunology, 69, 62-69.

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Hsp90-beta ELISA Quantification Protocol

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The levels of Hsp90-beta in serum and MPE were detected by a method of sandwich ELISA, which was originally developed using rabbit anti-human Hsp90-beta antibody. The test was performed according to the manufacturer’s instructions (Xitang Biotech, Shanghai, China) [10 (link), 14 (link)]. First, the 96-hole flexible micro titration plate (xitang biotechnology co., LTD., Shanghai, China) was coated with a serum (or MPE) sample diluted by PBS (pH 7.4) containing 1% BSA. After that, the hole was incubated with the monoclonal anti-Hsp90-beta antibody (10 ng/mL) at 37 °C for 1 h, then followed by reaction with avidin-conjugated peroxidase (Dako Cytomation) using a Substrate Reagent (R&D Systems) at 37 °C for 15 min. Finally, the chromogenic reaction was terminated by adding 50 μL 2 N sulfuric acid, and the intensity was measured at 450 nm wavelength by photometer. The standard curve of each plate was drawn using the concentration of the standard sample and the corresponding OD value of each hole.

Biaoxue R., Min L., Tian F., Wenlong G, & Hua L. (2018). Elevated Hsp90-beta contributes to differential diagnosis of pleural effusion caused by lung cancer and correlates with malignant biological behavior of lung cancer. BMC Pulmonary Medicine, 18, 188.

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