As a first screening of potential aptamers, FLAAs were performed as previously described34 (link) using an EnVision® 2105 multimode plate reader (PerkinElmer Inc., Waltham, USA). All reagents were diluted to their final concentrations in BP-T. Targets were immobilized either on Greiner FLUOTRAC™ 600 microplates (Merck KGaA, Darmstadt, Germany) for three hours at RT and 300 rpm followed by 14 h at 18 °C, or on Pierce™ Nickel Coated Plates (Thermo Fisher Scientific, Waltham, USA) for one hour at RT and 300 rpm. Wells were incubated with either 0.6 µg protein in PBS or with PBS only (negative control). Subsequently, wells were washed thrice with BP-T and incubated with 60–100 pmol of activated ssDNA for one hour at RT and 300 rpm. Micro titer plates (MTP) were washed thrice with BP-T and incubated with a 1:200 dilution of Quant-iT™ OliGreen™ ssDNA reagent (Thermo Fisher Scientific, Waltham, USA) for up to 20 min. Fluorescence was measured thrice (excitation 485 nm, emission 527 nm) after 12 min of dye incubation for clones from SR11.1 and 14.1 and after 20 min of dye incubation for clones from SR14.2_Mask. Measurements were performed in duplicate or triplicate. Fluorescence values were averaged, and negative control values were subtracted.
Pierce nickel coated plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce Nickel Coated Plates are a type of laboratory equipment used for various applications. They are made with a nickel coating, which provides a smooth and uniform surface. The core function of these plates is to serve as a platform for various experiments and procedures requiring a stable and consistent surface.
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Lab products found in correlation
Spectramax m5 plate reader, by Agilent Technologies (2 mentions)
Synergy neo2, by Agilent Technologies (2 mentions)
Quant it oligreen ssdna reagent, by Thermo Fisher Scientific (1 mentions)
Stop reagent for tmb substrate, by Merck Group (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
Rabbit anti ctb antibody, by Merck Group (1 mentions)
Goat anti rabbit igg hrp antibody, by Southern Biotech (1 mentions)
Amplex ultrared reagent, by Thermo Fisher Scientific (1 mentions)
Spectramax minimax 300 imaging cytometer, by Molecular Devices (1 mentions)
Glomax discover, by Promega (1 mentions)
Victor3, by PerkinElmer (1 mentions)
12 protocols using pierce nickel coated plate
1
Fluorescence-based Aptamer Affinity Screening
2
RNAPII-Drc.strep Binding Assay
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ELISA was performed in Pierce Nickel coated plates (Thermo Fisher Scientific). 100 pmol RNAPII, diluted in PBS with 2% bovine serum albumin (BSA; Sigma), was added to each well. After a 1h incubation, wells were washed three times with PBST (140 mM NaCl, 10 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3, 0.1% Tween) and three times with PBS. Drc.strep was then added at 100 pmol in PBS with 2%BSA followed by a 1 h incubation. The wash steps were repeated before adding a 1:5000 dilution of StrepMAB-Classic, HRP conjugate (IBA) in PBS + 2%BSA. After a final 1 h incubation, the reaction by HRP was started with 1-step Slow TMB-ELISA substrate solution (Thermo Fisher Scientific). Stop Reagent for TMB Substrate (Sigma) was added after 25 min and colorimetric detection was done at OD450 with a Bio-Rad model 680 microplate reader. All ELISA binding assays were performed in triplicate, RNAPII.strep served as positive control and negative controls included wells where RNAPII, Drc.strep or both were replaced by their respective buffer solution.
3
Evaluation of mAb Binding Kinetics
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Pierce Nickel Coated Plates (Thermo Fisher Scientific) were incubated overnight at 4°C with equivalent amount of sE2. The following day, a dilution series of mAbs or soluble receptor were incubated 1 hour at room temperature in two replicates of each dilution step. Plates were washed and binding was detected with horseradish peroxidase–conjugated anti-human IgG secondary antibody (Thermo Fisher Scientific).
4
Nickel-Coated Plates for His-tagged Protein Immobilization
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Pierce Nickel
Coated Plates (Thermo Fisher Scientific) were used to immobilize His-tagged
ECD-Her2 proteins (1 h incubation). After washing with PBS + 0.05%
Tween20, we performed a chemiluminescent assay as described above
for CMD-based patterned surfaces.
Coated Plates (Thermo Fisher Scientific) were used to immobilize His-tagged
ECD-Her2 proteins (1 h incubation). After washing with PBS + 0.05%
Tween20, we performed a chemiluminescent assay as described above
for CMD-based patterned surfaces.
5
Protein-Protein Interaction Mapping
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Bait proteins were bound to either Pierce® Nickel coated plates (15242; Thermo scientific) or ANTI-FLAG® high sensitivity, M2 coated plates (P2983, Sigma) by 2 h incubation at 37 °C. Unbound protein was removed by repeated washing and non-specific binding sites were blocked with 0.1–1% BSA/TBS. Bait-coated plates were incubated with prey proteins at 4 °C overnight. Binding of the prey protein to the coated plate was detected using primary antibodies raised against the prey proteins, combined with species matched HRP-conjugated secondary antibodies. Plates were read at 450 nm after HRP chromogenic substrate reaction using the TMB-peroxidase substrate system (KPL, Gaithersburg, MD, USA) according to manufacturer’s protocol. List of proteins and primary antibodies used can be found in Tables S2 and S3 .
6
Nanobody Screening Against Nitrated 14-3-3β
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The transformed TG1 cells were incubated with hyperphage (PRHYPE, Progen Biotechnik GmbH, Heidelberg, Baden- Wuerttemberg, Germany). The phage particles presenting the VHH library on their tips were collected. Phages containing the Nb fragments were enriched with solid phase panning. As a negative selection, Enzyme-Linked Immunosorbent Assay (ELISA) wells were coated with 5 µg of KLH at 4°C and phage particles were added to the wells and incubated at room temperature for 1.5 h. The unbound phage was moved to another round of negative selection for a total of three rounds. Afterwards, unbound phage were moved to positive selection with nitrated KLH. Bound phages were eluted with 0.1 M triethylamine and used for reinfection of TG1 cells, which were then used for one round of negative selection with WT 14-3-3ß bound by the (6x) His to Pierce® Nickel Coated Plates (ThermoScientific). The unbound phage were used for two subsequent panning rounds of positive sections with nitroTyr 14-3-3ß immobilized to the Ni-coated plates resulting two full length Nb sequences (Nb-G5 and Nb-F110).
7
Wheat Seed Protein Expression Analysis
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The total protein extracted from transgene positive wheat seeds tissues was then separated by SDS-PAGE on their molecular masses. The expression of vaccine antigen in seeds was verified by western blot analysis. The band were separated using 8% gel and were incubated with PBST containing 5% skimmed milk for one hour. The blot was then incubated with primary with 1:10000 ration with polyclonal rabbit anti-CTB antibody (Sigma-Aldrich, Inc,) over night at 4°C with continuous shaking. The blot was then washed three times with PBST and then was incubated with secondary goat anti-rabbit IgG-HRP antibody (Southern Biotechnology, Birmingham, AL) with ratio 1:5000 in PBST containing 5% skimmed milk. The expected bands corresponding to TM-1 protein was detected by substrate NBT/ BCIP and developed with X-Ray for 5 seconds exposure. The quantitation of specific band was performed using software ImageJ.
2.6 ELISA quantification Enzyme-linked immunosorbent assay (ELISA) was performed using Pierce Nickel Coated Plates (Thermo Fisher Inc.) to detect the expression of TM-1 recombinant vaccine in wheat seeds by following manufacture's instruction. All the plant samples were analyzed in duplicate. For detection of antibody titer in immunized chickens, chickens were bled, and the serum was analyzed by the ELISA against GM antibody (Idexx Laboratories) following manufacture's instruction.
2.6 ELISA quantification Enzyme-linked immunosorbent assay (ELISA) was performed using Pierce Nickel Coated Plates (Thermo Fisher Inc.) to detect the expression of TM-1 recombinant vaccine in wheat seeds by following manufacture's instruction. All the plant samples were analyzed in duplicate. For detection of antibody titer in immunized chickens, chickens were bled, and the serum was analyzed by the ELISA against GM antibody (Idexx Laboratories) following manufacture's instruction.
8
Nickel-Based Affinity Purification and Detection
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Cells (grown in 24 well plates) were lysed in lysis buffer (50 mM Tris-Hcl, 1M NaCl, 10 mM TCEP, 25 mM Imidazole, protease inhibitors) and processed as described above. Total protein (50 ug) was added to a single well on the PierceTM Nickel Coated Plates (Thermo Scientific) and incubated for 4–6 h at 4C. Each well was then gently washed 3 times with wash buffer (50 mM Tris-Hcl, 0.1M NaCl, 10 mM TCEP, 50 mM Imidazole). Each well was incubated with the anti-FLAG-M2-peroxidase antibody for 1 h at 4C. After washed each well 6 times with ice-cold PBS, the Amplex® UltraRed Reagent (Molecular Probes) assay solution was added (200 ul/well). Plates were incubated for 10-20 min at room temperature (in dark). 200 ul of the reaction was transferred to a 96well BLACK plate and read on the SpectraMax Minimax 300 Imaging Cytometer (Molecular Devices).
9
SARS-CoV-2 Spike Protein Binding Assay
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PierceTM Nickel Coated Plates (Thermo Fisher Scientific) were coated with pre- and postfusion stabilized F proteins (kindly gifted by J. McLellan [23 (link),24 (link)]) at a concentration of 1 µg/mL for 1 h at room temperature on a shaking plate. Coated plates were blocked with 1% bovine serum albumin (BSA) overnight and washed four times with PBS-Tween (PBS-T). 1:3 serial dilutions of ATAC-0025, palivizumab (pre- and postfusion F-specific mAb) and D25 (prefusion F-specific mAb) were incubated for 1 h at room temperature. Analyses were performed in triplicate. Next, plates were washed four times with PBS-T and incubated with goat anti-human HRP for palivizumab and D25 or goat anti-mouse HRP for ATAC-0025 (ThermoFisher Scientific) for 1 h at room temperature. After four final washes with PBS-T, 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma) was added to the plates and incubated at room temperature for 30 min. The colorimetric reaction was stopped with a stop solution (2 N sulfuric acid). Absorbance was measured at 450 nm using a spectrophotometer (GloMax Discover, Promega, Madison, WI, USA).
10
LOCKR Protein Interaction Assay
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His-tagged LOCKR was expressed per the above protocol from pET28b+ while the Key was expressed fused to superfolder GFP (sfGFP) without a his-tag in pET21b+. The his-tagged LOCKR was purified to completion and dialyzed into TBS (20 mM Tris, 150 mM NaCl, pH 8.0 at room temperature); the Key-GFP remained as lysate for this assay. 100 μL LOCKR at >1 μM was applied to a 96-well black Pierce® Nickel Coated Plate (ThermoFisher) and incubated at room temperature for 1 hour. Sample was discarded from the plate and washed 3x with 200 μL TBST (TBS + 0.05% Tween-20). 100 μL of lysate containing Key-GFP was added to each well and incubated at room temperature for 1 hour. Sample was discarded from the plate and washed 3x with 200 μL TBST (TBS + 0.05% Tween-20). The plate was washed 1x with TBS, and 100 μL of TBS was added to each well. sfGFP fluorescence was measured on a Molecular Devices SpectraMax M5 plate reader or BioTek Synergy Neo2 plate reader; fluorescence was measured at 485 nm excitation and 530 nm emission, with a bandwidth of 20 nm for excitation and emission.
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