HEK293T cells were transiently transfected with Flag-tagged M2 wild type (WT) and M2e Ala scan mutants. 24 h after infection the cells were detached, washed and blocked. Cells were stained with 20 μg/ml M2e VHH-23m or 20 μg/ml F-VHH-4 and subsequently fixed with 2% paraformaldehyde. After permeabilization (10 × permeabilization buffer diluted in double-distilled water; eBioscience), cells were stained with mouse anti-Histidine tag antibody (MCA1396, Abd Serotec) and rabbit anti-Flag tag antibody (F7425, Sigma-Aldrich). Binding of the primary antibodies was revealed with donkey anti-mouse IgG coupled to Alexa Fluor 647 (1/600; Invitrogen) and donkey anti-rabbit IgG coupled to Alexa Fluor 488 (1/600; Invitrogen). The median fluorescence intensity (MFI) of the cells was determined with an LSRII HTS flow cytometer (BD) and was calculated by subtracting the median fluorescence of binding of M2e-VHH-23m or F-VHH-4 to transfected cells from the median fluorescence of untransfected cells bound by M2e-VHH-23m or F-VHH-4.
Mca1396
Manufactured by Bio-Rad
Sourced in United Kingdom
The MCA1396 is a laboratory equipment product from Bio-Rad. It is a piece of hardware designed for specific laboratory functions. A detailed and unbiased description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Pe labeled goat anti mouse igg, by Jackson ImmunoResearch (3 mentions)
Facscanto 2, by BD (3 mentions)
Maxisorp microtiter plate, by Thermo Fisher Scientific (2 mentions)
A3562, by Merck Group (2 mentions)
Alexa fluor 594 labeled goat anti mouse antibody, by Thermo Fisher Scientific (2 mentions)
Imark microplate absorbance reader, by Bio-Rad (2 mentions)
Ap124p, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsr 2 hts flow cytometer, by BD (1 mentions)
F7425, by Merck Group (1 mentions)
Maxisorp 96 well microtiter plates, by Thermo Fisher Scientific (1 mentions)
Nxa931, by GE Healthcare (1 mentions)
A01861 200, by GenScript (1 mentions)
Streptavidin hrp, by BD (1 mentions)
Odyssey imager software, by LI COR (1 mentions)
Power blotter xl, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd goat, by LI COR (1 mentions)
Odyssey clx, by LI COR (1 mentions)
19 protocols using mca1396
1
M2e Epitope Mapping in HEK293T Cells
2
Nanobody Binding to Human PD-1 Cells
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Example 1
Binding to cell-expressed human PD-1 was evaluated on human PD-1 over-expressing CHO cells. A Nanobody dilution series was prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1×105 cells/well were transferred to a 96-well V-bottom plate and resuspended in 100 μL Nanobody dilution. After 30 minutes incubation at 4° C., the cells were washed with 100 μL/well assay buffer and resuspended in 100 μL/well of 1 μg/ml anti-FLAG (Sigma, F1804) or anti-HIS (AbD Serotec, MCA 1396). Samples were incubated for 30 minutes at 4° C., washed with 100 μL/well assay buffer, and resuspended in 100 μL/well of 5 μg/ml PE-labeled Goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071). Samples were incubated for 30 minutes at 4° C., washed, and resuspended in 100 μL/well of 5 nM TOPRO3 (LifeTechnologies, T3606) solution before analysis on FACS CANTO II (BD). The data from these experiments are set forth in
This Example demonstrated that the anti-human PD-1 monovalent Nanobody F023700706 bound to human PD-1 in a manner similar to the F023700275 monovalent Nanobody from which it was derived.
3
Quantifying CsgA Amyloid Fiber Assembly
4
Visualizing CsgA Fibers with Fluorescent Labeling
5
Antibody Raising and Detection
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Polyclonal rabbit antisera were raised against purified proPhoA or SecA (Davids Biotechnologie). For His-tagged proteins, mouse anti–His antibodies (MCA 1396; Serotec) were used. HRP-AP goat anti–rabbit IgG (111-035-0030) or HRP AP goat anti–mouse IgG (115-035-1460) secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc.
6
M2e-peptide ELISA for VHH Detection
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M2e-peptide ELISA was performed as described by De Filette et al.44 (link) Briefly, maxisorp 96-well microtiter plates (Thermo Fisher Scientific) were coated overnight with 100 ng M2e peptide (SLLTEVETPIRNEWGCRCNDSSD, corresponding to M2e of human H3N2 viruses) diluted in 0.1 M carbonate-bicarbonate buffer, pH 9.6. After overnight coating, wells were washed and blocked with 5% milk powder in phosphate buffered saline (PBS). Next, dilution series of the solution containing the VHHs (BALF or supernatant of cell culture) were added to the wells and incubated for 90 min. The bound VHHs were detected with mouse anti-histidine tag antibody (MCA1396, Abd Serotec) and horseradish peroxidase (HRP)-linked anti-mouse IgG (NXA931, GE Healthcare). Detection was done by adding 50 μL of TMB (Tetramethylbenzidine, BD OptETA) to every well; reactions were stopped by addition of 50 μL of 1M H2SO4. The absorbance was measured at 450 nm with an iMark Microplate Absorbance Reader (Bio Rad) using 655 nm as background measurement.
7
Human PD-1 Binding Nanobody Evaluation
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Example 1
Binding to cell-expressed human PD-1 was evaluated on human PD-1 over-expressing CHO cells. A Nanobody dilution series was prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1×105 cells/well were transferred to a 96-well V-bottom plate and resuspended in 100 μL Nanobody dilution. After 30 minutes incubation at 4° C., the cells were washed with 100 μL/well assay buffer and resuspended in 100 μL/well of 1 μg/ml anti-FLAG (Sigma, F1804) or anti-HIS (AbD Serotec, MCA 1396). Samples were incubated for 30 minutes at 4° C., washed with 100 μL/well assay buffer, and resuspended in 100 μL/well of 5 μg/ml PE-labeled Goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071). Samples were incubated for 30 minutes at 4° C., washed, and resuspended in 100 μL/well of 5 nM TOPRO3 (LifeTechnologies, T3606) solution before analysis on FACS CANTO II (BD). The data from these experiments are set forth in
This Example demonstrated that the anti-human PD-1 monovalent Nanobody F023700706 bound to human PD-1 in a manner similar to the F023700275 monovalent Nanobody from which it was derived.
8
Evaluating Anti-Human PD-1 Nanobody Binding
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Example 1
Binding to cell-expressed human PD-1 was evaluated on human PD-1 over-expressing CHO cells. A Nanobody dilution series was prepared in assay buffer: PBS/10% FBS/0.05% sodium azide. 1×105 cells/well were transferred to a 96-well V-bottom plate and resuspended in 100 μL Nanobody dilution. After 30 minutes incubation at 4° C., the cells were washed with 100 μL/well assay buffer and resuspended in 100 μL/well of 1 μg/ml anti-FLAG (Sigma, F1804) or anti-HIS (AbD Serotec, MCA 1396). Samples were incubated for 30 minutes at 4° C., washed with 100 μL/well assay buffer, and resuspended in 100 μL/well of 5 μg/ml PE-labeled Goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071). Samples were incubated for 30 minutes at 4° C., washed, and resuspended in 100 μL/well of 5 nM TOPRO3 (LifeTechnologies, T3606) solution before analysis on FACS CANTO II (BD). The data from these experiments are set forth in
This Example demonstrated that the anti-human PD-1 monovalent Nanobody F023700706 bound to human PD-1 in a manner similar to the F023700275 monovalent Nanobody from which it was derived.
9
ELISA Binding Assay for SARS and MERS Protein Interactions
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Wells of microtiter plates (type II, F96 Maxisorp, Nuc) were coated overnight at 4°C, respectively, with 100 ng recombinant MERS-CoV S-2P protein (with foldon), SARS-CoV-1 S-2P protein (with foldon), MERS-CoV RBD, MERS-CoV NTD, MERS-CoV S1, SARS-CoV-1 RBD, SARS-CoV-1 NTD or Fc-tagged SARS-CoV-2 RBD-SD1. The coated plates were blocked with 5% milk powder in PBS. Dilution series of the VHHs were added to the wells. Binding was detected by incubating the plates sequentially with either mouse anti-Histidine Tag antibody (MCA1396, Abd Serotec) followed by horseradish peroxidase (HRP)-linked anti-mouse IgG (1/2000, NXA931, GE Healthcare) or Streptavidin-HRP (554066, BD Biosciences) or by an HRP-linked rabbit anti-camelid VHH monoclonal antibody (A01861-200, GenScript). After washing 50 μL of TMB substrate (Tetramethylbenzidine, BD OptETA) was added to the plates and the reaction was stopped by addition of 50 μL of 1 M H2SO4. The absorbance at 450 nM was measured with an iMark Microplate Absorbance Reader (Bio Rad). Curve fitting was performed using nonlinear regression (Graphpad 7.0).
10
Visualizing CsgA Fibers with Fluorescent Labeling
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Mature CsgA fibers were deposited onto poly-L-lysine treated microscope slides51 (link) and slides were subsequently blocked for nonspecific binding by incubation with 5% (w/v) bovine serum albumin (BSA) for 10 min. The slides were then incubated at room temperature for 5 min with 3 μL 25 µM CsgC labeled with Alexa Fluor®488. Next, 10 μL of a mixture of an anti-6xHis antibody (AbD Serotec; MCA1396; 1:50 dilution) and an Alexa Fluor® 594-labeled goat anti-mouse antibody (Invitrogen; A11005; 1:20 dilution) in PBS was added for the staining of CsgA fibers. After 10 min incubation, slides were washed by flushing 3 times with 5 mL deionized water before examining them using a TE2000-U Nikon microscope.
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