Nunc Maxisorp polystyrene plates were coated with Spike Trimer (Excellgene, Monthey, Switzerland) or RBD (Excellgene, Monthey, Switzerland) at 1 μg/ml in PBS pH 7.4 (50 μl/well) overnight at 4°C; peptide RBM436-507 coating was at 2 μg/ml in Carbonate buffer, pH 9.6; 20-mers P11-P16 at 10 μg/ml in PBS, pH 7.4. After blocking for 1 hr at room temperature (RT) with PBS pH 7.4, BSA 3% (A4503 - Merck KGaA, Darmstadt, Germany), sera diluted 1/100 in PBS pH 7.4, BSA 1%, Tween-20 0.05% were incubated on the plate (50 μl/well) for 2 hours at RT. After 3 washings with PBS Tween-20 0.05% (150 μl/well), goat anti-human IgG HRP (A0293 - Merck) diluted 1:5000 in PBS BSA 1% Tween-20 0.05% was added to the plates at 50 μl/well and incubated for 2 hours. For IgM and IgA determination, goat anti-human IgM HRP conjugate (A0420 – Merck) or goat anti-human IgA HRP conjugate (A0295 - Merck) diluted 1:20,000 in PBS, BSA 1%, Tween 0.05% were added to the plates. After three washings with PBS Tween-20, 0.05%, enzymatic activity was measured at 450 nm after TMB addition (T4444 - Merck) and blocked by H2SO4 1M.
T4444
Manufactured by Merck Group
Sourced in United States
T4444 is a piece of laboratory equipment designed for conducting various scientific experiments and analyses. Its core function is to provide a controlled and consistent environment for conducting experiments or tests. The technical specifications and capabilities of this product are not available for an unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (2 mentions)
A8706, by Merck Group (2 mentions)
Goat anti human igg hrp, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Ab97240, by Abcam (1 mentions)
Anti 5hmc antibody, by Active Motif (1 mentions)
G9023, by Merck Group (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Ab1187, by Abcam (1 mentions)
Tween 20, by AppliChem (1 mentions)
Anti mouse antibodies coupled to hrp, by Southern Biotech (1 mentions)
Goat anti mouse total ig, by Southern Biotech (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dy582, by R&D Systems (1 mentions)
Dy485, by R&D Systems (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Bio-Rad (1 mentions)
Alpha hemolysin, by Merck Group (1 mentions)
Anti α hemolysin, by Merck Group (1 mentions)
11 protocols using t4444
1
SARS-CoV-2 Spike Protein Antibody ELISA
2
SEA-specific Antibody Detection ELISA
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SEA-specific serum antibodies were detected by homemade ELISA. Liver or intestinal SEA were diluted in carbonate buffer (1 ug/ml) and used for overnight coating of 96-well plates at 4°C. After washing with 0.05% Tween 20 in PBS, the wells were blocked with 1% BSA at 37°C for 2 hours. Then the wells were probed with diluted mouse serum (1:100) for 2 hours. After washing, the wells were incubated with horseradish peroxidaseconjugated goat anti-mouse IgG (1:8,000; Merck, #A2554) or IgG1 (1:2,500; Abcam, #ab97240) for 1.5 hours. Finally, the wells were repeatedly washed, and tetramethylbenzidine liquid substrate (Merck, #T4444) was used to visualize the reaction. The reaction was stopped by 1M HCl and read at 450 nm.
3
Quantification of 5-hydroxymethylcytosine
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The 96-well microtiter plate was coated with 10 pmol avidin (0.66 μg, SIGMA A8706) in 0.1 M NaHCO3. Biotin-5mC-DNA (IDT) substrates were then captured followed by incubation with TET2CD (0.4 µg) in 100 μl assay buffer [50 mM HEPES pH 6.5, 100 mM NaCl, 0.1 mM Fe(NH4)2(SO4)2 or FeCl3, and indicated concentration of AA along with 1 mM 2-OG] for 2 h at 37 °C. Reactions were stopped by adding 0.05 M NaOH (100 µL). After washing, wells were blocked with 2% BSA and probed with anti-5hmC antibody (Active motif, Cat# 39769, 1: 3000) at 4 °C overnight and visualized by HRP-conjugated anti-rabbit secondary antibody (Santa Cruz, Cat# sc-2004, 1:10,000) and developed by TMB (100 µl/well; SIGMA, T4444) and the color development was stopped by adding 50 µl of 2 M H2SO4 and measuring optical densities at 450 nm.
4
Quantitative ELISA for Neutrophil Proteins
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In all, 100 μl of neutrophil cell lysate (1–5 μg/μl protein in Laemmli buffer) was incubated in 96-well ELISA plate (maxisorb Nunc plate) for 2 h at RT to bind neutrophil proteins onto the plate surface. Wells were washed with 200 μl PBS/Tween (0.1%) and blocked by incubating with 200 μl PBS/Tween (0.1%) supplemented with 5% bovine serum albumin (BSA) and 5% goat serum (Sigma-Aldrich, G9023) for 1 h at RT. After washing the wells with 200 μl PBS/Tween (0.1%), 100 μl of rabbit anti-SPM antibody (Covalab, S.A.S., Villeurbanne, France, pab0022) or rabbit serum (Covalab) as negative control was given to the wells in 1 : 100 dilution in PBS/Tween (0.1%) supplemented with 5% BSA and 5% goat serum for 2 h at RT. Wells were washed 3x with 200 μl PBS/Tween (0.1%) and followed by secondary antibody (horseradish peroxidase (HRP)-conjugated anti-rabbit antibody) (Covalab, lab0273) in 1 : 1000 dilution in 100 μl PBS/Tween (0.1%) for 1 h at RT. Wells were washed 3x with 200 μl PBS/Tween (0.1%) and incorporated polyamines were detected using 100 μl substrate solution (3,3',5,5'-tetramethylbenzidine ready-to-use substrate solution, Sigma, T4444) for 5–10 min at RT. Reaction was determined by adding 50 μl 2 N sulfuric acid and optical density was measured at 450 nm by an ELISA reader.
5
Quantification of DNA 5-hydroxymethylation
6
Quantifying Collagen II in Scaffolds
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Collagen II was quantified as described elsewhere [50 (link)]. To determine the amount of collagen II in the scaffolds, 50 µL of the supernatant of the dissolved scaffolds (see Section 4.7 ) was used with a capture antibody mouse anti-chick collagen type II (1:2000, #7048; Chondrex, Woodinville, WA, USA) and a biotin-conjugated detection antibody mouse monoclonal anti-type II collagen (1:1000, # 7006, Chondrex) in an ELISA. Detection was performed using a streptavidin–horseradish peroxidase (# DY998; R&D Systems, Minneapolis, MN, USA) based conversion of the substrate 3,3′,5,5′-tetramethylbenzidine (#T4444; Sigma-Aldrich, St. Louis, MO, USA) and its photometric determination. The concentration was calculated using a calibration line of purified human collagen type II (# CC052; Millipore Sigma, Darmstadt, Germany).
7
Quantifying Importin-NLS Interactions
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The method was based on previously published microtiter plate assays47 (link). In brief, 96-well clear plates were coated with GST-NLS fusion proteins using bicarbonate/carbonate buffer pH 9.6 for 2 h at room temperature. The plates were washed using TBS containing 0.05% v/v Tween20 (TBST). Blocking was achieved using 5% (w/v) skim milk in TBST for 2 h at room temperature. Wells were washed three times in TBST buffer and incubated with decreasing concentrations of 6xHis-tagged importin αΔIBB (400, 300, 200, 100, 50, 25, 12.5 and 0 nM) diluted in TBS for 2 h. The plate was washed three times, blocked for 2 h, washed a further three times and then incubated for 2 h with 100 µL a 1/5000 dilution of anti-6xHis 4HRP conjugated rabbit polyclonal antibody (Abcam ab1187). The plate was washed a further three times before addition of 100 µL of TMB substrate (Sigma T4444). The colorimetric reaction proceeded for 20 min before being stopped with 100 µL of 2 M H2SO4, and the absorbance measured at 450 nm using an Epoch microplate spectrophotometer (Biotek). One-site specific binding analysis using least squares fit was performed using GraphPad Prism version 7.00 for Mac, GraphPad Software, La Jolla California USA, www.graphpad.com .
8
Measurement of Anti-SARS-CoV-2 Antibodies in Mice
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Anti-SARS-CoV-2 Ig responses were measured in mouse sera using enzyme-linked immunosorbent assays (ELISA). Nunc MaxiSorp™ plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated directly with Monomeric soluble RBD (Institute of Protein Design) at 1.25 µg/mL in 1× PBS overnight at 4 °C, and blocked for 1 h with PBS-t, PBS containing 0.05% (v/v) Tween 20 (Applichem, Boca Raton, FL, USA), and 2% (w/v) bovine serum albumin (BSA) (Sigma, Tokyo, Japan). The plates were washed with PBS-t and incubated with 4× serial dilutions of mouse serum samples diluted 1/100 in PBS containing 0.05% (w/v) BSA. Plates were incubated for 1 h at 37 °C, washed, and then incubated for 1 h at 37 °C with anti-mouse antibodies coupled to HRP (Southern Biotech, Birmingham, AL, USA) including goat anti-mouse total Ig, IgG1, IgG2b, or IgG2c, an allelic form of IgG2a expressed in C57BL/6 mice [20 (link),21 (link)] at 1:6000 dilution. Plates were then washed before the addition of TMB supersensitive (Sigma, T4444) substrate solution. The reaction was stopped at 7 min by adding 1M sulfuric acid. Absorbance was directly measured at 450 nm using a microplate reader (Biotek Instruments, Winooski, VT, USA).
9
Quantitative ELISA Assay Protocol
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Supernatants of exposed cells were collected and diluted 1:1 in ELISA dilution buffer according to manufacturer’s protocol (R&D; DY582, DY206-05, and DY485). Briefly, plates were coated with capture antibody for 18 h, blocked for 1 h, washed 3× and exposed for 2 h to 100 μl diluted supernatant from exposed cells. Then plates were washed 3× and detection antibody was added for another 2 h after which the plates were washed again for 3× and streptavidin-HRP was added for 30 min. Plates were washed again and HRP substrate (Sigma; T4444) was added for 10–30 min after which the reaction was stopped by adding 50 μl of 1 M H2SO4. Colorimetric determination was performed in an ELISA plate reader at 450 nm wavelength.
10
Enzyme-Linked Immunosorbent Assay for Alpha-Hemolysin
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A 96-well plate (Nunc, MaxiSorp) was coated O/N, at 4 °C, with 100 µl anti-α-hemolysin (S7531, Sigma Aldrich) diluted 1:12 500 (V/V) in sodium carbonate buffer (0.05 M, pH: 9.5). Wells were then blocked for 2 h, RT, using 250 µl BSA (1 %, W/V) in wash buffer. Patient samples and alpha-hemolysin spiked serum samples, diluted 1:10 (V/V) in PBS, were subsequently added to the wells in duplicates (100 µl/well) and incubated for 2 h. anti-α-hemolysin (S7531, Sigma Aldrich) diluted 1:12 500 (V/V) were added (100 µl/well) for an additional 2 h before secondary HRP-conjugated goat anti-rabbit-antibodies (Bio-Rad, 1721019), diluted 1:4000 (V/V) in wash buffer, was finally added (1 h, RT). Wells were incubated for 10–15 min with substrate 3,3′,5,5′-Tetramethylbenzidine (TMB, Sigma Aldrich, T4444) (100 µl/well) before stop solution in the form of 100 µl H2SO4 (1 M) was added and the absorbance was read at 450 nm. Between each steps, wells were washed 3 times using 0.05 % Tween 20 in PBS (V/V). The absorbance values recorded from the alpha-hemolysin spiked serum samples were used to draw a calibration curve which was then used for the quantification of alpha hemolysin in patient samples.
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