Ab50370

Manufactured by Abcam

Ab50370 is a laboratory equipment product. It is a specific protein detection tool designed for use in scientific research applications.

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Lab products found in correlation

Infinite 200 rx plate reader, by Tecan (3 mentions) 96 well flat bottom plate, by Thermo Fisher Scientific (2 mentions) Tetramethylbenzidine tmb substrate, by Thermo Fisher Scientific (2 mentions) Maxisorp, by Thermo Fisher Scientific (2 mentions) Ab171522, by Abcam (1 mentions) Maxisorp flat bottom 96 well plate, by Thermo Fisher Scientific (1 mentions)

5 protocols using ab50370

Phage Display Binding Assay Protocol

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A Nunc Maxisorp flat-bottom 96-well
plate was coated with 6 × 10⁹ pfu GV per well (in
TBS, pH 8) and incubated overnight at 4 °C. Plates coated with
2% (w/v) bovine serum albumin (BSA) were used as negative controls.
The next day, the plates were blocked with 2% (w/v) BSA at room temperature
for 1 h, shaking at 800 rpm. The plates were then washed with 0.1%
TBST (3 × 1 min) before adding 20 μL of amplified phage
from each biopanning cycle to each well in 5% (w/v) BSA. After further
incubation at room temperature for 1 h, shaking at 800 rpm, the plates
were washed with 0.5% TBST (3 × 5 min) before adding 100 μL
of horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody
(Abcam ab50370, diluted 1:500) and incubating at room temperature
for 1 h, shaking at 800 rpm. After further washes in 0.5% TBST (3
× 5 min), we added 100 μL of the tetramethylbenzidine (TMB)
substrate (Thermo Fisher Scientific) to each well. The plates were
incubated in the dark for 10 min, and the absorbance was measured
at 370 nm using an Infinite 200 Rx plate reader (Tecan Life Sciences)
with 25 flashes in 96-well flat-bottom plate mode.

Chan S.K., Zhao Z., Penziner S., Khong E., Pride D., Schooley R.T, & Steinmetz N.F. (2022). Isolation of a Peptide That Binds to Pseudomonas aeruginosa Lytic Bacteriophage. ACS Omega, 7(42), 38053-38060.

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Phage ELISA for Antigen-Specific Enrichment

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The phage ELISA was used to assess the enrichment of antigen-specific phage particles. GFP (or iRFP713) (100 ng) was coated per well at 4°C overnight and then blocked with 5% skimmed milk-PBS at RM for 3 h. The phage particles amplified after each round of panning were diluted into 2 × 10¹⁰ phages in 100 µl of 3% skimmed milk-PBS and then incubated in wells at RM for 2 h. Anti-M13 mAb HRP conjugate (Abcam, ab50370) diluted 1:3,000 in 5% skimmed milk-PBS was added and then incubated at RM for 1 h. The ELISA was developed with TMB substrate (Abcam, ab171522) and then read at 450 nm.

Wei L., Wang M., Xiang H., Jiang Y., Gong J., Su D., Al Azad M.A., Dong H., Feng L., Wu J., Chan L.L., Yang N, & Shi J. (2021). Bamboo Shark as a Small Animal Model for Single Domain Antibody Production. Frontiers in Bioengineering and Biotechnology, 9, 792111.

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Screening Fab Fragments via Phage ELISA

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All ELISA experiments were performed in 96-well plates coated with 50 μl of 2 μg ml⁻¹ neutravidin in Na₂CO₃ buffer, pH 9.6 and subsequently blocked by 0.5% BSA in PBS. A single-point phage ELISA was used to rapidly screen the binding of the obtained Fab fragments in phage format. Colonies of E. coli XL1-Blue harbouring phagemids were inoculated directly into 500 μl of 2xYT broth supplemented with 100 μg ml⁻¹ ampicillin and M13-KO7 helper phage. The cultures were grown at 37 °C for 16–20 h at 280 r.p.m. in a 96-deep-well block plate. Culture supernatants containing Fab phage were diluted tenfold in PBST buffer. After 15 min of incubation, the mixtures were transferred to ELISA plates that were incubated with 50 nM biotinylated trimeric G_i1 in experimental wells and with buffer in control wells for 15 min. The ELISA plates were incubated with the phage for another 15 min and then washed with PBST. The washed ELISA plates were incubated with horseradish perixoidase (HRP)-conjugated anti-M13 mouse monoclonal antibody (ab50370, Abcam,1:5,000 dilution in PBST) for 30 min. The plates were again washed, developed with TMB substrate and then quenched with 1.0 M HCl, and the absorbance at 450 nm was determined. The background binding of the phage was monitored by the absorbance from the control wells.

Kang Y., Kuybeda O., de Waal P.W., Mukherjee S., Van Eps N., Dutka P., Zhou X.E., Bartesaghi A., Erramilli S., Morizumi T., Gu X., Yin Y., Liu P., Jiang Y., Meng X., Zhao G., Melcher K., Ernst O.P., Kossiakoff A.A., Subramaniam S, & Xu H.E. (2018). Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature, 558(7711), 553-558.

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High-throughput Phage Display Screening

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We coated each well of a Nunc Maxisorp flat-bottom 96-well plate with 10 μg TMV (in 0.1 M bicarbonate buffer, pH 8.6) and incubated overnight at 4 °C. Plates coated with 5% (w/v) BSA was used as negative controls. Next day, plates were blocked with 5% (w/v) BSA and incubated at room temperature for 1 h, shaking at 800 rpm. The plates were then washed with 0.1% TBST (3 × 1 min). We added 20 μL of amplified phage from each biopanning cycle to each well in 5% (w/v) BSA and incubated at room temperature for 1 h, shaking at 800 rpm. Plates were then washed with 0.5% TBST (3 × 5 min) followed by the addition of 100 μL HRP-conjugated anti-M13 monoclonal antibody (Abcam ab50370, diluted 1:500) and incubation at room temperature for 1 h, shaking at 800 rpm. After further washes in 0.5% TBST (3 × 5 min) we added 100 μL of the tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) to each well. The plates were incubated in the dark for 10 min and the absorbance was measured at 370 nm using an Infinite 200 Rx plate reader (Tecan Life Sciences) with 25 flashes in 96-well flat-bottom plate mode.

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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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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