Experiments were performed at room temperature in a recording chamber containing 1 ml of 50% Neurobasal/50% Hibernate low fluorescence solution (Brain Bits, Springfield, IL) supplemented with B27 (Sigma). Postsynaptic endplates were identified by the application of a non-blocking AChR antibody, mAb35 (Table 1 ) conjugated to an Alexa Fluor546 fluorchrome using a Monoclonal Antibody Labeling Kit (Invitrogen) for 1 hour prior to the recording session. NMJs were identified by the expression of GFP in apposition to mAb35 fluorescence identified using a CCD camera coupled to an Olympus upright fluorescence microscope (Centre Valley, PA). Images were captured using a Nikon digital camera. Micropipettes used for recordings had tip resistances between 10 and 50 MΩ and were filled with 3 M KCl. Reponses were recorded with a Sutter amplifier and processed with Clampex 10.2 software (Molecular Devices). All data were analyzed using MiniAnalysis (Synaptosoft, Decatur, GA). Quantal contents were determined by the direct method (m = spontaneous endplate potential/miniature endplate potential; sEPP/mEPP) using the mean mEPP as determined following application of 2.5 μM TTX. In some cases, 5 μM μ-conotoxin GIIIB (Alomone Labs, Jerusalem, Israel) was added to the recording solution to block Na+ channel-mediated myotube contraction.
Monoclonal antibody labeling kit
Manufactured by Thermo Fisher Scientific
The Monoclonal Antibody Labeling Kit is a laboratory product designed to facilitate the covalent conjugation of a label or reporter molecule to a monoclonal antibody. The kit provides the necessary reagents and instructions to enable the efficient and reproducible labeling of antibodies for various applications, such as immunoassays, flow cytometry, and immunohistochemistry.
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Lab products found in correlation
Upright fluorescence microscope, by Olympus (1 mentions)
Clampex 10, by Molecular Devices (1 mentions)
Digital camera, by Nikon (1 mentions)
μ conotoxin giiib, by Alomone (1 mentions)
Propidium iodide solution, by Thermo Fisher Scientific (1 mentions)
Flat bottomed 96 well tissue culture plates, by Corning (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
Alexa 647 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa488 conjugated annexin 5, by Thermo Fisher Scientific (1 mentions)
Rabbit anti v5, by Merck Group (1 mentions)
Rabbit anti chmp4b, by Abcam (1 mentions)
Rabbit anti actin, by Merck Group (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
5 protocols using monoclonal antibody labeling kit
1
Quantification of Neuromuscular Junction Signaling
2
Anti-TF mAb and ADC Binding Assay
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To analyze the affinity and internalization of the anti‐TF mAbs and ADCs, the mAbs and ADCs were directly conjugated with Alexa647 using a monoclonal antibody labeling kit (Invitrogen) according to manufacturer's instruction. The affinities of the mAbs and the ADCs against pancreatic cancer cells were analyzed by flow cytometry. Inside a 2 ml tubes, 2 × 105 harvested cells were incubated with 0.2 μg of each Alexa647‐conjugated mAbs and ADCs for 30 min at 4°C. After washing with PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA (B. E. PBS), the cells were nuclear stained with a propidium iodide (PI) solution (Invitrogen, Eugene, OR). The stained cells were analyzed by flow cytometry using Guava easyCyte (Millipore, Billerica, MA).
To analyze the internalization of the anti‐TF ADC into cancer cells, 3 × 103 cells of BxPC‐3 were pre‐cultured in flat‐bottomed 96‐well tissue culture plates (Corning, Corning, NY) overnight. Then, 0.2 μg of Alexa647‐conjugated ADC were added to the cells and incubated at 37°C for 0 and 3 hr. To identify the lysosomes, the cells were also incubated for 1 hr with 50 nM of LysoTracher (Invitrogen). After rinsing with PBS, the cells were fixed with 4% paraformaldehyde (Wako) for 10 min and then nuclear stained with a 4′6‐diamidino‐2‐phenylindole‐2HCl (DAPI) solution (Roche, Basel, Switzerland).
To analyze the internalization of the anti‐TF ADC into cancer cells, 3 × 103 cells of BxPC‐3 were pre‐cultured in flat‐bottomed 96‐well tissue culture plates (Corning, Corning, NY) overnight. Then, 0.2 μg of Alexa647‐conjugated ADC were added to the cells and incubated at 37°C for 0 and 3 hr. To identify the lysosomes, the cells were also incubated for 1 hr with 50 nM of LysoTracher (Invitrogen). After rinsing with PBS, the cells were fixed with 4% paraformaldehyde (Wako) for 10 min and then nuclear stained with a 4′6‐diamidino‐2‐phenylindole‐2HCl (DAPI) solution (Roche, Basel, Switzerland).
3
Fluorescent Conjugation of Anti-HER2 Antibodies
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Anti-HER2 antibodies (trastuzumab, 39S and anti-HER2-Bs) were labeled with Alexa Fluor 647 (AF647) using Monoclonal Antibody Labeling Kit (Cat# A20186, Life Technologies) according to the manufacturer’s protocol. Before labeling, the buffer of the antibodies was replaced with phosphate buffered saline PBS using Zeba Spin Desalting Columns, 40K MWCO (Cat# 87768, Thermo Scientific, Waltham, MA, USA). The concentration of the antibodies was adjusted to 1 mg/mL with PBS. One hundred micrograms of antibody was used in each labeling reaction. After labeling, antibody concentration and the degree of labeling were determined by measuring A280 and A650 following the manufacturer’s protocol. The labeling ratio of mole of Alexa Fluor dye per mole of antibody was between 5.6 and 8.4. The Alexa Fluor-conjugated antibodies were stored at 4 °C for 1–2 months.
4
Antibody Binding to Immobilized Peptides
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Specific binding of the antibody anti-M7 to the immobilized peptides M7 (EQLKKSKTL), or the control peptides scrambled sM7 (KLSLEKQTK), or the peptide M8 (EEFRIHFT)41 (link) was tested in a solid-phase binding with immobilized peptides in 96-well ELISA plates (Nunc). Binding of anti-M7 was detected by addition of biotinylated anti-mouse IgG and subsequent color reaction after the incubation with HRP-coupled streptavidin and TMB-substrate. Specific binding was calculated by subtracting the binding of mouse IgG to the peptides. Alternatively, binding of anti-M7 was tested in western blot on cell lysates from native CHO cells or CHO cells stably transfected with human Mac-1. Full western blots are shown in Supplementary Figure 1 . To test binding of the antibody anti-M7 to human leukocytes, anti-M7 was labeled with Alexa Fluor-647 according to the manufacturer’s protocols (Monoclonal Antibody Labeling Kit, Life Technologies). Human leukocytes were isolated from healthy donors by centrifugation and Red Blood Cell lysis, and stimulated with PMA (200ng/ml), incubated with anti-M7-Alexa 647 (1 μg and 5 μg) and antibody binding was quantified by flow cytometry.
5
Antibody Utilization for Immunoprecipitation and Imaging
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Antibodies used for immunoprecipitation and immunolabeling include rabbit anti-BIN1 exon 13 (custom made, generous gift from Sarcotein), mouse anti-BIN1 exon 17 (clone 99D, Sigma), recombinant monoclonal anti-BIN1 exon 13 (generous gift from Sarcotein), chicken anti-GFP (Abcam), mouse anti-Flag (Sigma), rabbit anti-CHMP4B (Abcam), rabbit anti-V5 (Sigma), rabbit anti-actin (Sigma), mouse and rabbit anti-GST (Santa Cruz), mouse anti-CD63 (Thermo Scientifics), fluorescein-conjugated anti-PI(4,5)P2 IgM (Echelon Bioscience), and fluorescein-conjugated anti-PI(3,4,5)P3 IgM (Echelon Bioscience). Alexa 647-conjugated WGA, Alexa 488-conjugated annexin V, Alexa 555-conjugated annexin V, Alexa 647-conjugated annexin V, and Alexa 647-conjugated phalloidin were purchased from Life Technologies. For imaging and flow cytometry, recombinant anti-BIN1 exon 13 was conjugated with Alexa 647 using a monoclonal antibody labeling kit (Life Technologies).
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