The 96-well plates were coated with mCD248-ECD-Fc (400 ng) in PBS overnight (4 °C), blocked (PBS 3% (w/v) BSA), then MMRN2FL transfected HEK293T lysates (6 × 106 cells/ml) added diluted in PBS (1:50). CLEC14A-ECD-Fc (200 ng) or anti-His antibodies (200 ng), then for CLEC14A-ECD-Fc, C2 (400 ng) was incubated. Binding detected with anti-mouse-HRP (1:5000), visualized using BM Blue POD substrate (Roche).
Bm blue pod substrate
Manufactured by Roche
Sourced in Germany, Switzerland, United States
The BM Blue POD Substrate is a laboratory reagent used for the detection and quantification of enzyme activity. It is a chromogenic substrate that undergoes a color change upon enzymatic reaction, allowing for the measurement of enzyme levels in various samples. The core function of the BM Blue POD Substrate is to provide a standardized and reliable method for the colorimetric analysis of peroxidase-like enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin conjugated horseradish peroxidase, by Roche (3 mentions)
Anti pkm2 antibody, by Cell Signaling Technology (2 mentions)
Pv3832, by Thermo Fisher Scientific (2 mentions)
Immuno module, by Thermo Fisher Scientific (2 mentions)
Recombinant pkm2, by Abcam (2 mentions)
Vivaspin 6 columns, by Sartorius (2 mentions)
Jam a elisa kit, by Sino Biological (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Ab151230, by Abcam (1 mentions)
A6154, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Spectramax m2 plate reader, by Molecular Devices (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti fibronectin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti α sma antibody, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Abcam (1 mentions)
Goat anti rabbit hrp conjugate antibody, by Bio-Rad (1 mentions)
18 protocols using bm blue pod substrate
1
MMRN2 Binding to CLEC14A Quantification
2
Methyltransferase Activity Assay for METTL3-METTL14
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Methyltransferase activities of wild-type and mutant METTL3-METTL14 complexes were measured using an antibody-based assay. Assay conditions were derived from a previously reported radioactivity-based assay (Li et al., 2016 (link)) and modified to ensure optimal turnover within the timescale of the assay. Reactions mixtures contained 200 nM 3’-biotinylated RNA substrate (5’-UACACUCGAUCUGGACUAAGCUGCUC-3’) and 1 μM S-adenosyl methionine in 20 mM Tris buffer at pH 8.0. The reactions were initiated by the addition of 100 nM protein and incubated at 37°C for 180 min. To detect the production of methylated RNA, reaction mixtures were transferred to the 96-well neutravidin coated plates (Pierce) and incubated for 60 min at 4°C. Following extensive washing and blocking, the plate was incubated first with a m6A-specific primary antibody (Abcam ab151230, used at 1:400 dilution), and subsequently with horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, A6154, used at 1:5000 dilution). m6A antibody binding was quantified by measuring the conversion of a colorimetric substrate 3,3’,5,5’-tetramethylbenzidine (TMB, supplied as BM Blue POD substrate by Roche Diagnostics GmbH, Germany) at a wavelength of 390 nm. Reactions lacking the SAM cofactor were performed to measure the background signal for each enzyme construct due to non-specific antibody binding.
3
Quantification of FAK-PKM2 Interaction
4
Purification and Detection of C426 Avimer
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The bacterial lysates were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the His-tag at the N-terminal end, the C426 avimer protein was purified by Ni-NTA affinity chromatography His-Bind Resin according to the manufacturer’s instructions, and stored at −20°C. ELISA analysis was conducted to detect expression of protein using anti-His antibody ELISA kit (cat. no. AKR-130; USA). The 96 well micro plates were coated with 100 μl/well of the coating solution (Sodium carbonate 50 mM, pH=9.6) and were incubated at 4°C overnight. The wells were washed five times using washing solution [20 ml PBS (1×) +20 μl Tween 20], and then were kept at 37°C for two hr. After adding the 100 μl of Anti-His6-Peroxidase solution (10 mu/ml) per well, they were incubated at 25°C for an hr. Wells were washed again and treated with 100 μl/well of BM Blue POD Substrate (Roche, Germany), and were incubated at 25°C until sufficient color was developed. To stop the color development, 100 μl/well of 2N sulfuric acid was added and then their absorbance was determined at 450 nm.
5
ELISA for HIV-1 Env Antibody Titers
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ELISA’s were performed following standard procedure. 2HB plates (96-well; Nalgene Nunc, Rochester, NY) were coated overnight with 90 ng/well HIV-1 Env-CN54 protein, diluted in 1X coating buffer (Cat # 6247ImmunoChemistry Technologies, Bloomington, MN), then washed with buffer (150 mM NaCl, 0.1% Tween-20), blocked for 1 hour in 5% nonfat milk, 3% heat-inactivated goat serum and 0.2% Tween-20 in PBS and then washed again. Serum samples were serially diluted in blocking buffer, and then added to the wells in duplicate and incubated for 2 hours at 37 °C. The plates were then washed and HRP-conjugated anti-mouse IgG (Thermo Scientific) was added at 1:2500 dilution and incubated for 1 hour at 37 °C. After washing, the plates were developed with 3,3′–5,5′-tetramethylbenzene (BM Blue POD substrate; Roche Applied Science, Indianapolis, IN); the reaction was stopped by addition of 1M H2SO4 and the optical density was read at 450 nm using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, Ca). The reported titers correspond to the reciprocal of the highest serum dilution showing a three times higher OD value than background.
6
Quantifying Chemokine Release in Supernatant
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The release of human CX3CL1 and CXCL16 into the supernatant was analyzed by ELISA. Before the measurement, the culture supernatants were concentrated from 3 ml to 0.5 ml using Vivaspin 6 columns (10.000 MWCO) (Sartorius, Göttingen, Germany). 96 well plates were coated in PBS overnight at 4°C with a capture antibody against CX3CL1 (4 μg/ml) or CXCL16 (1 μg/ml), respectively. Before adding the samples, unspecific binding to the plate was blocked with washing buffer (PBS + 0.05% Tween) containing 2% BSA for 2 h at room temperature. Samples were incubated for another 2 h at room temperature, and the bound chemokines were detected with a biotinylated secondary antibody against CX3CL1 (0.3 μg/ml) or CXCL16 (0.5 μg/ml). The chromogenic reaction was mediated by a standard procedure using 0.1 U/ml streptavidin-conjugated horseradish peroxidase (Roche, Basel, Switzerland) and the BM Blue POD substrate (Roche).
7
Glioma Tumor Tissue Western Blot Analysis
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Glioma tumor tissue and adjacent normal tissue were washed with ice-cold PBS, homogenized on ice, and lysed by protein lysate (Pierce). After centrifugation, the protein concentration was measured by BCA protein assay kit (Pierce). Fifty micrograms of lysate was used for western blot. Briefly, whole cell protein extracts were separated on 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Amersham). The blocked membrane was incubated first with anti-DKC1 polyclonal mouse antibody (Santa Cruz) or anti-beta-actin antibody (Sigma) and then with rabbit anti-mouse antibody conjugated with horseradish peroxidase (Sigma). The signal was detected using BM Blue POD Substrate (Roche). The following antibodies were used: anti-CTNNBIP1 antibody (1:200; Santa Cruz), anti-β-Catenin (t-β-Catenin) antibody (1:200; Product code: ab47426; Abcam), anti-β-Catenin (phospho T41 + S45, p-β-Catenin) antibody (1:200; Product code: ab81305; Abcam), anti-α-SMA antibody (1:200; Sigma, St. Louis, MO), and anti-fibronectin antibody (1:200; Santa Cruz). Anti-β-actin antibody (1:1000; Sigma) was used as a loading control.
8
Quantitative Measurement of Citrullinated Histone H3
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Citrullinated histone H3 (citH3) was measured as described24 (link), with minor modifications. Streptavidin-coated plates were incubated with anti-histone biotin (Roche) for 120 minutes. Plates were washed and incubated with plasma samples or standard (citH3, Cayman) for 90 minutes. After washing, anti-histone H3 (1:2000 in PBS + 1% bovine serum albumin [BSA], Abcam) was added for 60 minutes. Plates were washed and incubated with goat anti-rabbit HRP conjugate antibody (1:5000 in PBS + 1% BSA, BioRad) for 60 minutes, washed, and developed with BM Blue POD Substrate (Roche) for 20 minutes. The reaction was stopped by addition of 2 M H2SO4 and optical density was measured on a Promega GloMax Discover microplate reader (450 nm, reference 620 nm).
Double-stranded deoxyribonucleic acid (dsDNA) was quantified using the Quant-iT PicoGreen dsDNA Assay (Invitrogen) according to manufacturer´s instructions. Fluorescence was measured on a GloMax Discover microplate reader (Promega).
Double-stranded deoxyribonucleic acid (dsDNA) was quantified using the Quant-iT PicoGreen dsDNA Assay (Invitrogen) according to manufacturer´s instructions. Fluorescence was measured on a GloMax Discover microplate reader (Promega).
9
Quantifying AREG Release in ADAM17 Depleted HaCaT Cells
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To test effect of ADAM17 depletion on release of human amphiregulin (AREG) HaCaT cell were transfected with control or ADAM17-specific siRNA. Two days later, the medium was exchanged to medium without FCS and 24 hr later the supernatants were collected, centrifuged on 15,000 g for 5 min at 4°C, supplemented with cOmplete protease inhibitor cocktail and processed for analysis. Samples were tested with Human Amphiregulin DuoSet ELISA kit (DY262) from R and D Systems according to the manufacturer’s instructions. As substrate solution BM Blue POD Substrate from Roche was applied. The measurement was performed at 450 nm and the correction wavelength at 540 nm.
10
ELISA for scFv43M2 binding to HPV16-E7 and HPV18-E7
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Methods to produce, purify, and characterize recombinant HPV16-E7, HPV18-E7, and the 43M2 scFv have been already described.14 (link) ELISA was carried out as published with minor modifications. Briefly, recombinant HPV16-E7 and HPV18-E7 proteins were adsorbed onto Maxisorp microtiter plates (NUNC, Thermofisher Scientific) overnight at 4°C. Afterwards, wells were saturated with 2% non-fat dry milk (Bio-Rad) at 37°C for 2 hrs, and then incubated at 37°C overnight with 3 µg/mL of purified scFv43M2 followed by 1:3000 dilution of anti-flag M2 mAb (Sigma) for 1 hr. Wells were then washed, and a 1:20,000 dilution of anti-mouse HRP-conjugated polyclonal IgG (SBA Inc.) was added for 40 mins. After washing, the antigen-antibody complexes were detected by tetramethylbenzidine (BM blue, POD substrate, Roche). After 30 mins, the enzymatic reaction was stopped, and optical density (OD) measured at 450 nm.
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