Maxisorp flat bottom 96 well plate

Manufactured by Thermo Fisher Scientific

Sourced in Denmark

The MaxiSorp flat-bottom 96-well plates are a type of laboratory equipment used in various experimental procedures. They are designed with a flat bottom to provide a stable surface for sample placement and analysis. The plates are made of high-quality materials to ensure consistent and reliable performance.

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Lab products found in correlation

Goat anti rabbit igg hrp, by Jackson ImmunoResearch (2 mentions) Power wave 200 microplate scanning spectrophotometer, by Agilent Technologies (2 mentions) Ph d 12 phage display peptide library kit, by New England Biolabs (1 mentions) Fluostar optima, by BMG Labtech (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions) Dulbecco phosphate buffered saline (dpbs), by R&D Systems (1 mentions) Recombinant human upa, by R&D Systems (1 mentions) Ab50370, by Abcam (1 mentions) Infinite 200 rx plate reader, by Tecan (1 mentions) Tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions) Versamax 190 microplate reader, by Molecular Devices (1 mentions) Goat anti rat hrp, by Santa Cruz Biotechnology (1 mentions) Donkey anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions) Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions) Hrp conjugated rabbit anti human igg antibody, by Agilent Technologies (1 mentions) Superblock blocking buffer, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated goat anti mouse igm, by Jackson ImmunoResearch (1 mentions) Peroxidase conjugated donkey anti rabbit igg secondary antibody, by Jackson ImmunoResearch (1 mentions) Phosphate buffered saline (pbs), by Serva Electrophoresis (1 mentions)

Close spelling variants of this product

96-well plates (1169 citations) Maxisorp plates (569 citations) MaxiSorp (425 citations) MaxiSorp flat-bottom plates (6 citations) Maxisorp 96-well flat bottom plates (4 citations) Maxisorp flat-bottom 96-well ELISA plates (4 citations) Nunc MaxiSorp® flat-bottom 96 well ELISA plates (3 citations)

23 protocols using maxisorp flat bottom 96 well plate

Enzyme-linked Immunosorbent Assay for Affinity Determination

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96-well plates (MaxiSorp^® flat-bottom 96-well plate, Fisher Scientific Inc., Fair Lawn, NJ) were coated with 500 ng actin in 100 μl carbonate-bicarbonate buffer (pH 9.5) for 1 h at 37°C. The plate was washed once with TBS and then blocked with 3% BSA in 200 μl TBS buffer at 37°C, for 1 h. Wells were then washed once with 250 μl TBS. trT2-50 was added at 1:2 dilutions in 100 μl TBS, starting from 500 ng/well, incubated for 1 h at 37°C and then washed three times with TBS containing 0.1% Tween-20 (TBST). Each well was then treated with 100 μl rabbit anti-trT2-50 diluted 1:500 in TBS and incubated for 1 h at 37°C. The wells were washed three times with TBST and then incubated with 100 μl goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, West Grove, PA) diluted 1:10,000 in TBS, for 1 h at 37°C. Wells were then washed twice with TBST, and once with TBS before 100 μl substrate (1-step Ultra TMB-ELISA, Pierce, Fisher Scientific Inc.) were added. Absorbance at 655 nm was measured 10 min thereafter using a Power Wave 200 Microplate Scanning Spectrophotometer (Bio-Tek Instrument, Winooski, VT). Affinity was evaluated by double reciprocal plot [28 , 29 (link)].

Nesiel-Nuttman L., Schwartz B, & Shoseyov O. (2014). Human recombinant truncated RNASET2, devoid of RNase activity; A potential cancer therapeutic agent. Oncotarget, 5(22), 11464-11478.

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Actin Binding Affinity Assay

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96-well plates (MaxiSorp® flat-bottom 96-well plate, Fisher Scientific Inc., Fair Lawn, NJ) were coated with 500 ng actin in 100 μl carbonate-bicarbonate buffer (pH 9.5) for 1 h at 37°C. The plate was washed once with TBS and then blocked with 3% BSA in 200 μl TBS buffer at 37°C, for 1 h. Wells were then washed once with 250 μl TBS. trT2-50, trT2-50m or each of the 29 peptides were added at 1:2 dilutions in 100 μl TBS, starting from 500 ng/well, incubated for 1 h at 37°C and then washed three times with TBS containing 0.1% Tween-20 (TBST). Each well was then treated with 100 μl rabbit anti-trT2-50 diluted 1:500 in TBS and incubated for 1 h at 37°C. The wells were washed three times with TBST and then incubated with 100 μl goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, West Grove, PA) diluted 1:10,000 in TBS, for 1 h at 37°C. Wells were then washed twice with TBST, and once with TBS before 100 μl substrate (1-step Ultra TMB-ELISA, Pierce, Fisher Scientific Inc.) were added. Absorbance at 655 nm was measured 10 min thereafter using a Power Wave 200 Microplate Scanning Spectrophotometer (Bio-Tek Instrument, Winooski, VT). Affinity was evaluated by double reciprocal plot [36 ,37 (link)].

Nesiel-Nuttman L., Doron S., Schwartz B, & Shoseyov O. (2014). Human RNASET2 derivatives as potential anti-angiogenic agents: actin binding sequence identification and characterization. Oncoscience, 2(1), 31-43.

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Phage Display Screening for TMV Binders

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TBPs were isolated using a PhD-12 Phage Display Peptide Library Kit (New England Biolabs) as described previously with slight modifications.^{[8 (link)]} We coated each well of a Nunc Maxisorp flat-bottom 96-well plate with 10 μg TMV and incubated plates overnight at 4 °C. Four rounds of affinity selection were carried out to enrich for TBPs by increasing the stringency of selection in each round. This was achieved by increasing the concentration of Tween-20 in TBST from 0.1% to 0.3%, 0.4% and then 0.5%. The enriched phages were eluted and amplified according to the manufacturer’s protocol.

Chan S.K, & Steinmetz N.F. (2022). Isolation of Tobacco Mosaic Virus-Binding Peptides for Biotechnology Applications. Chembiochem : a European journal of chemical biology, 23(11), e202200040.

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P-selectin Ligand Expression Assay

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To evaluate the P-selectin ligand expression on AsPC-1, Capan-2, and MIA PaCa-2 cells, respectively, 0.3 µg recombinant human P-selectin Fc Chimera Protein in 50 µL DPBS (R&D systems) was immobilized in each well of a Nunc MaxiSorp flat-bottom 96-well plate for 12 h at 4 °C. Afterwards, unspecific binding sites were blocked with 250 µL BSA solution (4% in DPBS) per well for 4 h and wells were rinsed with 250 µL DPBS thrice. Tumor cells were detached with EDTA (0.2 g/L EDTA × 4 Na, Sigma Aldrich) for 10 min at 37 °C, labeled with Calcein-AM (1 µM Calcein-AM concentration) for 1 h, and rinsed twice with warm DPBS buffer. Then, 5 × 10⁵ tumor cells in 100 µL DPBS were added in each well and the plate was gently shaken on a plate shaker for 2 h. Unbound cells were removed with DPBS and adherent cells were lysed with 1% Triton X-100 (v/v) in DPBS (Merck). Calcein-AM was quantified in a FLUOstar Optima plate reader (BMG Labtech, Ortenberg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Haschemi R., Gockel L.M., Bendas G, & Schlesinger M. (2021). A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells. International Journal of Molecular Sciences, 22(7), 3323.

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Binding Specificity of MLK SH3 Domains

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To determine whether MIP and NS5A can cross-react with other members of the MLK subfamily, SH3 domains of MLK1, MLK2, and MLK4 were purified as GST fusion proteins as described above. To determine their binding properties, 100 μl (≈2 μm) of biotinylated peptides, MIP (AIRINPNGTWSRQAETVES-K-biotin), MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin; as negative control), and NS5A (KKAPTPPPRRRR-GGG-K-biotin), were first captured in wells of a Nunc MaxiSorp flat-bottom 96-well plate coated with NeutrAvidin (100 μl, 7.5 μg/ml) and then blocked with 2% skim milk in PBS. The GST-MLK1 SH3, GST-MLK2 SH3, GST-MLK2 SH3, and GST-MLK4 SH3 domain fusion proteins (0.15 μm) were incubated with each of the target peptides, MIP, NS5A, or MIP-R12A, for 1–1.5 h. The rest of the assay was performed as described above for IC₅₀ determination of MIP.

Kokoszka M.E., Kall S.L., Khosla S., McGinnis J.E., Lavie A, & Kay B.K. (2018). Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3. The Journal of Biological Chemistry, 293(35), 13553-13565.

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Competitive Binding Assay for NS5A and MIP

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Competition ELISA was performed to determine whether MIP and NS5A peptides compete in binding the MLK3 SH3 domain and to calculate the relative binding strength (i.e. IC₅₀) of the NS5A peptide. Biotinylated NS5A peptide (KKAPTPPPRRRR-GGG-K-biotin; 100 μl, ≈ 2 μm) was captured in wells of a Nunc MaxiSorp flat-bottom 96-well plate, which had been coated with NeutrAvidin (100 μl, 7.5 μg/ml), and blocked with 3% skim milk in PBS. Separately, the GST-MLK3 SH3(43–104) domain fusion protein (5 μg/ml) was preincubated (1–1.5 h) with increasing concentration (0.02–100 μm) of unlabeled MIP as competitor and then allowed to interact for 1 h with biotinylated MIP prebound on the ELISA plate (Fig. S7). The assay was then completed as described above for IC₅₀ determination of MIP.

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Isolation of uPA-Inhibiting Antibodies

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A fully human naïve Fab phage display library was used to identify inhibitory antibodies against human active uPA (30 (link)). Recombinant Human uPA (R&D Systems) was immobilized overnight in wells of a MaxiSorp® flat-bottom 96 well plate (Nunc) at 20 μg/mL in PBS (137 mM NaCl, 2.7 mM KCl, Na₂HPO₄, 10 mM, KH₂PO₄ 2 mM pH 7.4). The panning was accomplished in four rounds as described previously (31 (link), 32 (link)). After four rounds of selection, Fab was produced from 192 individual clones in a 96-well format, the Fabs that leaked into the cell culture media were screened for binding to uPA by ELISA. Clones with a positive signal in ELISA were analyzed by BstNI restriction analysis to identify the unique clones. Clones with unique sequence were expressed, purified, and tested for inhibition of uPA.

LeBeau A.M., Sevillano N., Markham K., Winter M.B., Murphy S.T., Hostetter D.R., West J., Lowman H., Craik C.S, & VanBrocklin H.F. (2015). Imaging Active Urokinase Plasminogen Activator in Prostate Cancer. Cancer research, 75(7), 1225-1235.

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Phage Display Selection of CPMV Binders

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5 μg of CPMV (in 0.1 M bicarbonate buffer, pH 8.6) was coated into each well of a Nunc Maxisorp flat-bottom 96-well plate and incubated overnight at 4 °C. 2% (w/v) BSA was also coated to serve as negative control. The next day, the plate was blocked with 2% (w/v) BSA and incubated at rt with shaking at 800 rpm for 1 h. Subsequently, the plate was washed thrice with 0.1% TBST for 1 min. 100 μL of amplified phage from the last biopanning cycle was added into each well followed by incubation at rt with shaking at 800 rpm for 1 h. Later, the plate was washed thrice with 0.5% TBST for 5 min. 100 μL of 1:500 dilution of HRP-conjugated anti-M13 monoclonal antibody (Abcam ab50370) was added and incubated at rt for 1 h with shaking at 800 rpm. The plate was again washed thrice with 0.5% TBST for 5 min followed by adding 100 μL of tetramethylbenzidine (TMB) substrate (Thermo Scientific Pierce) into each well. The plate was incubated in the dark for 10 min, and the absorbance was measured at 370 nm by using an Infinite 200 Rx plate reader (Tecan Life Sciences) with 25 flashes in 96-well flat-bottom plate mode. Monoclonal phages with at least 0.3 difference in absorbance value between CPMV and BSA were subjected for DNA sequencing (Eurofins Genomics).

Chan S.K, & Steinmetz N.F. (2021). Isolation of Cowpea Mosaic Virus-Binding Peptides. Biomacromolecules, 22(8), 3613-3623.

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CPMV Quantification by ELISA

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Two-fold serial dilutions of CPMV (40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, and 0 μg) were coated in a Nunc Maxisorp flat-bottom 96-well plate, and ELISA was performed similarly to the “monoclonal ELISA” with one modification: 10⁹ pfu of phage was added into each well for binding.

Chan S.K, & Steinmetz N.F. (2021). Isolation of Cowpea Mosaic Virus-Binding Peptides. Biomacromolecules, 22(8), 3613-3623.

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Quantifying Drosophila Insulin-Like Peptides

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Twenty 7–11-day old males were decapitated and placed in a 0.2 mL PCR tube with a hole punctured in the bottom. The tube was placed into another collection tube and centrifuged at 5000 rpm for 3 min at 4 °C. The extracted hemolymph was diluted with 100 μL cold PBS. 45 μL of the diluted hemolymph was coated on a MaxiSorp flat-bottom 96 well plate (Nunc) overnight at room temperature. The plate was incubated in block (0.02M NaPO₄ buffer pH 7.4, 150 mM NaCl, 1.27 mM EDTA, 1% BSA) for 1 hour at room temperature. The plate was washed twice with 0.05% Tween-20 in PBS then incubated with either rat anti-dILP2 (1:1000) or rabbit anti-dILP5 (1:2000) for 2 hours at room temperature. After three washes the plate was incubated with goat anti-rat HRP (1:2500, Santa Cruz) or donkey anti-rabbit HRP (1:2500, Santa Cruz) secondary antibody for 1 hour at room temperature. The plate was washed three times then incubated with 1X TMB ELISA substrate solution (eBioscience) for 15 min. The reaction was stopped with 1M phosphoric acid and the absorbance at 450 nm was measured using a VersaMax 190 Microplate Reader (Molecular Devices).
dILP2 and dILP5 levels were normalized to protein levels measured by BCA Protein Assay (Pierce). Anti-dILP2 and anti-dILP5 antibodies were provided by P. Leopold [26] (link).

Trinh I., Gluscencova O.B, & Boulianne G.L. (2018). An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion. Molecular Metabolism, 19, 13-23.

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