anti egfr biotin  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

doi: 10.1101/2023.07.19.549702

Figure Lengend Snippet: a, Immunological synapse formation between a representative CD4 + T cell expressing CD19-specific CARs and ZAP70 labeled with GFP (rainbow LUT), and a Raji cell (magenta) stained for CD20 by antibodies conjugated to ATTO643. b, A second example of synapse formation between a CD4 + CD19-specific CAR-T cell and a Raji cell. An increase in fluorescence signal of GFP channel (rainbow LUT) can be seen at the cell-cell junction confirming ZAP70 accumulation at the cell-cell contact site. c , 3D volume rendering of a CD4 + CD19-specific CAR-T cell and a Raji cell forming an immunological synapse. Scattered accumulation of GFP signal (white pointers) can be visualized at the contact site. d, Immunological synapse formation between a representative CD4 + T cell (rainbow color scale) without CAR construct expressing ZAP70-GFP and a Raji cell (magenta). Synapse formation was induced by addition of staphylococcal enterotoxin type E (SEE). e, No immunological synapse formation is seen between CD4 + T cells without CAR construct expressing ZAP70-GFP and Raji cells. f, Side view of a CD4 + T cell without CD19-CAR at the site of synapse formation (induced by addition of SEE) shows accumulation of ZAP70-GFP signal resembling the classical ‘bull’s eye’ organization. g, Scattered clustering of ZAP70-GFP at multiple foci (indicated with white arrows) interleaved by lower fluorescence intensity regions unlike the classical organization can be seen at the site of synapse formation for a CD4 + CD19-specific CAR-T cell (right panel). Scale bars, 5 µm.

Article Snippet: Prior to functional testing, EGFRt-positive T cells were enriched using biotin-conjugated anti-EGFR mAb and anti-biotin beads (Miltenyi Biotec), and subsequently expanded using a rapid expansion protocol based on CD3-TCR-activation via OKT-3 (Miltenyi Biotec) and irradiated donor-mismatched PBMCs (all CAR-T cells, unless indicated) or irradiated feeder cells expressing CD19 or anti-CAR surface-proteins (LV/SB comparison of CD19 and ROR2 CAR-T cells) for 7-14 days.

Techniques: Expressing, Labeling, Staining, Fluorescence, Construct

Journal: Journal of visualized experiments : JoVE

Article Title: Quantification of protein phosphorylation using single molecule pull-down

doi: 10.3791/63665

Figure Lengend Snippet: (A) Channel registration is first performed on images acquired from the nanogrid. After cropping the two spectral channels of interest (here, green and far-red), the fiducial images for each channel are overlaid (left). Enlargement of the box in the left image (Inset) shows that the images are not yet truly registered. The emitters in each channel are then fit to a Gaussian model and localized (Registration). Localization of emitters is shown as circles for the far-red channel and crosses for the green channel. The final step is to apply a local weighted mean transform to shift the far-red channel localization coordinates into the green channel reference frame (Aligned). The calculated local weighted mean transform is then used to register the subsequent SiMPull data. (B) Representative images of the green/EGFR-GFP channel and the far-red/AF647-anti-PY channel. Single emitters above the background photon count are identified and marked with boxes. (C) The emission profile within each selected box is fit to a Gaussian model and the emitters that fit the model of a single fluorophore PSF are kept. (D) A mask is created from the GFP emitters to identify location of EGFR-GFP (green). Colocalization of EGFR-GFP and AF647-anti-PY identifies phosphorylated receptors (white). (E) The fraction of phosphorylated receptors is calculated from the colocalized EGFR-GFP and AF647-anti-PY fits. Bar graph compares PV+EGF treatment to resting cells, averaged for multiple measurements. Error bars represent standard error calculated assuming a binomial distribution. Scale bars, 2 μm.

Article Snippet: Anti-Human EGFR (External Domain) – Biotin , Leinco Technologies, Inc , E101 , .

Techniques:

Journal: Journal of visualized experiments : JoVE

Article Title: Quantification of protein phosphorylation using single molecule pull-down

doi: 10.3791/63665

Figure Lengend Snippet: (A) From left to right, the first three panels are representative images of the autofluorescence on glass under the respective conditions: after piranha etching, with PEG, and PEG plus NaBH4 treatment (indicated with +). Additionally, surface functionalization is retained after NaBH4 treatment as demonstrated by minimal non-specific PY99-AF647 binding, while retaining robust binding of EGFR-GFP from the lysate. (B) To ensure optimal antibody labeling, a saturation curve should be acquired for each batch of antibody used. This figure shows the concentration curve for labeling EGFR with the site specific phosphotyrosine antibody, anti-EGFR-pY1173. Minimal phosphorylation is detected in untreated cells (Resting, gray diamond). As a control for non-specific binding, cells were also treated with the EGFR kinase inhibitor Lapatinib before addition of 100 nM EGF (magenta triangle), which shows the expected prevention of EGFR phosphorylation. Error bars represent standard error assuming a binomial distribution. (C) Fixation of the sample with a combination of PFA and GA prevents antibody dissociation over time. Error bars represent standard error assuming a binomial distribution. (D) False positives are excluded by selecting the appropriate threshold for Gaussian fitting. Comparing the histogram of fit intensities at a low threshold (Threshold = 0; top) between background (no lysate) and real data (plus cell lysate) allows for selection of appropriate value (Threshold = 475; bottom) to remove fits from autofluorescent spots in the green channel. Vertical magenta line indicates 475 photon threshold. Histograms are calculated from the same number of ROIs for each sample type (n=3). Scale bars, 2 μm.

Article Snippet: Anti-Human EGFR (External Domain) – Biotin , Leinco Technologies, Inc , E101 , .

Techniques: Binding Assay, Antibody Labeling, Concentration Assay, Labeling, Phospho-proteomics, Control, Selection

Journal: Journal of visualized experiments : JoVE

Article Title: Quantification of protein phosphorylation using single molecule pull-down

doi: 10.3791/63665

Figure Lengend Snippet: Table of Materials

Article Snippet: Anti-Human EGFR (External Domain) – Biotin , Leinco Technologies, Inc , E101 , .

Techniques: Electron Microscopy, Lysis, Recombinant, Bicinchoninic Acid Protein Assay, Convection, Protease Inhibitor, Plasmid Preparation, Antibody Labeling, Conjugation Assay, Binding Assay, Microscopy, Cell Counting, Transferring, Staining, Software