Journal: bioRxiv

Article Title: Rewarding capacity of optogenetically activating a giant GABAergic central-brain interneuron in larval Drosophila

doi: 10.1101/2022.12.19.521052

Figure Lengend Snippet: Confocal maximum projection images of stainings for mCD8::GFP, Syt1::SNAP, and TLN::CLIP , driven by the APL-specific intersectional driver APLi ( ; ) at the following developmental times: (A-A’’’) third-instar larva (L3); (B-C’’’) 6h after puparium formation (6h APF: calyx: B-B’’’; lobes: C-C’’’); (D-D’’’) 12h APF; (E-F’’’) adult (calyx: E-E’’’; lobes: F-F’’’). Brains were stained with a polyclonal chicken anti-GFP antibody to label the APL neuron (A-F). To label pre-synapses (A’-F’) and post- synapses (A’’-F’’), the pre-synaptic reporter synaptotagmin was fused to the chemical tag SNAPm (Syt1-SNAPm), and the post-synaptic reporter telencephalin was fused to CLIPm (TLN-CLIPm), respectively . Merged images are shown in (A’’’-F’’’). In the third-instar larva, pre-synaptic staining was largely restricted to the calyx (A’), whereas post- synaptic staining was distributed in both the calyx and the lobes (A’’). At 6h APF, both pre- and post-synaptic staining are similarly distributed to that in the larvae (B’-C’’’); notably, pre- synaptic structures seem to be more punctate (B’, C’), and fewer post-synaptic structures are detectable (B’’, C’’). As late as 12h APF, both pre- and post-synaptic structures are still detectable (D’, D’’); post-synaptic structures appear to be detached from the neurite (D’’, D’’’; yellow arrowhead). In adults, both pre- and post-synaptic markers are detectable in both the calyx and the lobes (E-F’’’). The data were acquired with a 40x oil objective; scale bars: 20 μm.

Article Snippet: The chemical substrates were SNAP- tag ligands (SNAP surface 549 - BG 549 [NEB, S9112S]) and CLIP-tag ligands (CLIP surface 647 - BC 647 [NEB, S9234S]) at final concentrations of 1 μM in 0.3% PBT.

Techniques: Staining

Journal: bioRxiv

Article Title: Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex

doi: 10.1101/2023.01.31.526478

Figure Lengend Snippet: The interaction between WDR76 and SPIN1 was characterized using Acceptor-Photobleaching Fluorescence Resonance Energy Transfer (apFRET) ( A-C ) and Fluorescence Cross-Correlation Spectroscopy (FCCS) ( D-E ). ( A ) Example image and intensity measurement of apFRET. Halo-WDR76 was labeled with HaloTag TMRDirect ligands and SNAP-SPIN1 was labeled with SNAP-Cell 505-Star ligands. ( B ) Averaged FRET efficiencies measured for Halo-WDR76 and SNAP-SPIN1 in live HEK293FRT cells. SNAP tag was used as donor in the control experiment to be tested with Halo-WDR76. Error bars stand for standard error of mean and p-values were calculated using two-tailed t-test. ( C ) FRET efficiencies measured for control proteins in live HEK293FRT cells. Separate Halo tag and SNAP tag were used as a negative control pair and a fusion protein of Halo tag and SNAP tag was used as positive control. Error bars stand for standard error of mean and p-values were calculated using two-tailed t-test. ( D ) Auto and cross-correlation curves for Halo-WDR76 and SNAP-SPIN1, and for Halo-WDR76 and SNAP tag as control experiment. ( E ) The average percentage of Halo-WDR76 binding to SNAP tag or SNAP-SPIN1 was calculated from the y amplitudes of correlation curves. Error bars stand for standard error of mean. Values was obtained based on the fitted correlation curves, t-test was not performed for this data.

Article Snippet: Halo tags were stained with HaloTag® Ligands TMRDirect (Promega), and SNAP tags were stained with SNAP-Cell® 505-Star (NEB).

Techniques: Fluorescence, Förster Resonance Energy Transfer, Spectroscopy, Labeling, Two Tailed Test, Negative Control, Positive Control, Binding Assay

Journal: bioRxiv

Article Title: Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex

doi: 10.1101/2023.01.31.526478

Figure Lengend Snippet: ( A ) Identification of proteins involved in non-homologous DNA end joining (NHEJ). Each dNSAF value plotted is the average from three biological replicates; error bars represent standard deviations. ( B ) Examples of time course imaging. In each experiment, a SNAP-tagged protein was co-expressed with Halo-WDR76. Halo-tagged proteins were labeled with HaloTag TMRDirect ligands and SNAP-tagged proteins were labeled with SNAP-Cell 505-Star ligands. Cells were also stained with HOECHST 33258. 405nm UV laser damage was introduced at timepoint 0 in a stripe-shaped confined area. ( C ) Recruitment of Halo- or SNAP-tagged proteins to laser induced damages. Recruitment level is presented by relative intensity at the stripe, calculated from the intensity of each timepoint normalized to the intensity before microirradiation. For Halo-WDR76, the sample co-expressed with SNAP-Control is plotted. Each data point is an average from all measured cells; error bars stand for standard error of mean. ( D ) The maximum recruitment of each protein within 2min after microirradiation. Each maximum recruitment value is an average from all measured cells; error bars stand for standard error of mean.

Article Snippet: Halo tags were stained with HaloTag® Ligands TMRDirect (Promega), and SNAP tags were stained with SNAP-Cell® 505-Star (NEB).

Techniques: Imaging, Labeling, Staining

Journal: ACS Omega

Article Title: Click Chemistry-Generated Auristatin F–Linker–Benzylguanine for a SNAP-Tag-Based Recombinant Antibody–Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells

doi: 10.1021/acsomega.2c06844

Figure Lengend Snippet: Protein validation/characterization of IMAC-purified 1711(scFv)-SNAP. (A) SDS-PAGE gel of purified and concentrated 1711(scFv)-SNAP. (B) 1711(scFv)-SNAP was subjected to a Western Blot analysis. A primary anti-his rabbit antibody (1:1000) was used, followed by a secondary goat anti-rabbit HRP antibody conjugate (1:5000). (C) Fluorescent blot and corresponding 10% SDS-PAGE gel of 1711(scFv)-SNAP conjugated with BG-Alexa Fluor 488 at different (protein: fluorophore) ratios 4:1, 2:1, 1:1 and 1:2.

Article Snippet: Commercially available BG-modified fluorophores, SNAP-Surface BG-Alexa Fluor 488 (BG-Alexa 488), and SNAP-Surface BG-Alexa Fluor 647 (BG-Alexa 647), purchased from New England Biolabs are photostable fluorescent substrates used to label SNAP-tag fusion proteins in solution or in live cells.

Techniques: Purification, SDS Page, Western Blot

Journal: ACS Omega

Article Title: Click Chemistry-Generated Auristatin F–Linker–Benzylguanine for a SNAP-Tag-Based Recombinant Antibody–Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells

doi: 10.1021/acsomega.2c06844

Figure Lengend Snippet: Cytotoxicity of 1711(scFv)-SNAP-AuriF toward EGFR-overexpressing tumor cells. (A) Fluorescent blot and associated 10% SDS-PAGE gel of 1711(scFv)-SNAP and 1711(scFv)-SNAP-AuriF (generated after 2, 3, and 4 h incubation) conjugated to BG-Alexa 647. (B, C) XTT viability assay was used to assess cytotoxicity following a 72 h incubation with 1711(scFv)-SNAP-AuriF on (B) MDA-MB-468 and (C) A2058 cells. IC 50 values relative to untreated cells were calculated using GraphPad Prism v5 software. Three biological repeats were made with duplicate treatments. Means and standard deviations are presented for each concentration.

Article Snippet: Commercially available BG-modified fluorophores, SNAP-Surface BG-Alexa Fluor 488 (BG-Alexa 488), and SNAP-Surface BG-Alexa Fluor 647 (BG-Alexa 647), purchased from New England Biolabs are photostable fluorescent substrates used to label SNAP-tag fusion proteins in solution or in live cells.

Techniques: SDS Page, Generated, Incubation, Viability Assay, Software, Concentration Assay

Journal: ACS Omega

Article Title: Click Chemistry-Generated Auristatin F–Linker–Benzylguanine for a SNAP-Tag-Based Recombinant Antibody–Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells

doi: 10.1021/acsomega.2c06844

Figure Lengend Snippet: Binding of 1711(scFv)-SNAP on South African breast cancer patients’ tissue sections and the pooled mean fluorescence intensities of EGFR expression. Using the LSM confocal 880 microscope, FFPE tissue sections were imaged. The mean of each patient’s fluorescent intensity data was extracted and tabulated for comparison. (A) 1711(scFv)-SNAP conjugated to BG-Alexa488 labels the cell membrane. (B) The corresponding bright field panel displaying cell morphology. (C) The DAPI panel showing nuclear staining of cells. (D) Merged panel of A-C. The 1711(scFv)-SNAP pooled label data for patients, whereby patient means were compared using an ANOVA. The mean intensity data indicated significant differences between all tumour and non-tumour tissues in the selected patient samples. Qualitative differences are indicated as a comparison of the fluorescence image panels of patient 2 (E-G) and patient 9 (H-J). These samples were normalized against an autofluorescence control for each patient. Differences in mean fluorescent densities between tumour and non-tumour of selected patients 2, 3, 7, 9, and 10 (K).

Article Snippet: Commercially available BG-modified fluorophores, SNAP-Surface BG-Alexa Fluor 488 (BG-Alexa 488), and SNAP-Surface BG-Alexa Fluor 647 (BG-Alexa 647), purchased from New England Biolabs are photostable fluorescent substrates used to label SNAP-tag fusion proteins in solution or in live cells.

Techniques: Binding Assay, Fluorescence, Expressing, Microscopy, Staining

Journal: PLoS ONE

Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins

doi: 10.1371/journal.pone.0073255

Figure Lengend Snippet: CD20 plasmid constructs were transiently transfected into HEK293 cells, grown for 22h before analysis in flow cytometry. A. Staining with a CD20 antibody, grey solid line; pSNKL-Q construct, black solid line; pSNKL-SNAP construct, dashed black line; non-transfected cells. B. same as A but stained with anti-HA antibody. C. same as A but with stained with the SNAP labeling reagent. D. CD20 in pSNKL-CD24. Black solid line; stained with CD20 antibody, grey solid line; stained with CD24 antibody, black dashed line; stained with HA antibody.

Article Snippet: For SNAP tag staining we used SNAP surface Alexa fluor 488 (NEB).

Techniques: Plasmid Preparation, Construct, Transfection, Flow Cytometry, Staining, Labeling

Journal: BMC Biotechnology

Article Title: An efficient protocol for the purification and labeling of entire yeast septin rods from E.coli for quantitative in vitro experimentation

doi: 10.1186/1472-6750-13-60

Figure Lengend Snippet: Purification of recombinant septin rods from E. coli . A . Coomassie stained SDS-PAGE of the purified septin rod. B . Isoelectric focussing of purified septin rods (left panel) or SNAP tagged septin rods (right panel). C . PAGE separation of the excised band from the IEF for SNAP tagged septin rods. All four septins are present in the complex. D . Analytical size exclusion chromatography of purified septin rods. The rods elute in one prominent peak containing all four constituents. The single septins can be detected by Western blot using antibodies against Cdc3 and the attached 6his tag and S tag, respectively.

Article Snippet: Purified SNAP tagged septin rods (ligand protein) were covalently labeled with BG-Biotin (New England Biolabs) by SNAP tag chemistry in MSP buffer (10 mM MES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) adjusted to 300 mM NaCl.

Techniques: Purification, Recombinant, Staining, SDS Page, Size-exclusion Chromatography, Western Blot

Journal: BMC Biotechnology

Article Title: An efficient protocol for the purification and labeling of entire yeast septin rods from E.coli for quantitative in vitro experimentation

doi: 10.1186/1472-6750-13-60

Figure Lengend Snippet: Fluorescence microscopy of SNAP tagged, TMR labeled septin filaments. Left panel: No filaments are formed under high salt conditions. Right panel: Filaments in low salt conditions.

Article Snippet: Purified SNAP tagged septin rods (ligand protein) were covalently labeled with BG-Biotin (New England Biolabs) by SNAP tag chemistry in MSP buffer (10 mM MES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) adjusted to 300 mM NaCl.

Techniques: Fluorescence, Microscopy, Labeling

Journal: BMC Biotechnology

Article Title: An efficient protocol for the purification and labeling of entire yeast septin rods from E.coli for quantitative in vitro experimentation

doi: 10.1186/1472-6750-13-60

Figure Lengend Snippet: Bni5 interacts with septins. A . Spin down assay: SDS-PAGE analysis of supernatant and pellet fraction after centrifugation of a sample containing Bni5-SNAP together with septin filaments (low salt (LS); left panel) or together with septin rods (high salt (HS); right panel). Bni5-SNAP co-sedimentation with the septins indicates interaction. B . Validation of the spin down assay with Western blot after SDS-PAGE using an anti-his antibody. 6his tagged Bni5-SNAP is exclusively detected in the pellet with the septins (6his-Cdc12 as control).

Article Snippet: Purified SNAP tagged septin rods (ligand protein) were covalently labeled with BG-Biotin (New England Biolabs) by SNAP tag chemistry in MSP buffer (10 mM MES, 150 mM NaCl, 0.05% Tween 20, pH 7.4) adjusted to 300 mM NaCl.

Techniques: Spin Down Assay, SDS Page, Centrifugation, Sedimentation, Western Blot