Proteins were expressed from the pMal-c2 vector (NEB), except for full length hnRNPA1 and related mutants, which were cloned into a modified pet11a vector (Novagen)(see Supplemental Experimental Procedures). Proteins were expressed in E. coli BL21(DE3) and purified with Ni-NTA and/or amylose resin. SNAP-PTB-IDRs were further purified through a Superdex200 column (GE Healthcare). Proteins were fluorescently-labeled with SNAP-Surface 488 or SNAP-Surface 649 (NEB) according to the manufacturer’s protocols. Amino acid residues of all proteins and nucleotide sequences of all RNAs are listed in Table S2 .
Snap surface 488
Manufactured by New England Biolabs
Sourced in United States
SNAP-Surface 488 is a fluorescent labeling system for SNAP-tag fusion proteins. The SNAP-tag is a protein that can be fused to a target protein and covalently labeled with a fluorescent dye. SNAP-Surface 488 contains the 488 nm fluorescent dye and is designed to be used for labeling SNAP-tag fusion proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pmal c2 vector, by New England Biolabs (1 mentions)
Snap surface 649, by New England Biolabs (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Naloxone, by Bio-Techne (1 mentions)
Morphine, by Merck Group (1 mentions)
Furimazine, by Promega (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Rhodamine 6g, by Merck Group (1 mentions)
Anti ps375, by Cell Signaling Technology (1 mentions)
Compound 101, by Hello Bio (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Pitstop 2, by Abcam (1 mentions)
Snap surface 549, by New England Biolabs (1 mentions)
Snap cell 647 sir, by New England Biolabs (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Snap cell tmr star, by New England Biolabs (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
12 protocols using snap surface 488
1
Purification and Labeling of Proteins
2
DAMGO Receptor Binding Assay
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D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin was obtained from Mimotopes. Morphine, Rhodamine 6G and M2-anti-FLAG were from Sigma-Aldrich (Gillingham, Dorset, United Kingdom). Naloxone was from Tocris. SNAP-Surface 488 was from New England Biolabs (Ipswich, MA, United States). The antibody anti-pS375 was from Cell Signaling. Secondary antibodies (raised in donkey) conjugated to Alexa-Fluor 488 or 647 were from Jackson ImmunoResearch. Coelenterazine h was from NanoLight. Furimazine was from Promega. Compound 101 was from HelloBio. Pitstop2 was from Abcam. PTx was from Millipore.
3
SNAP-tag Protein Labeling
4
Rapamycin-induced SNAP-tag Detection
5
SNAP-Tag Labeling for Liposome Orientation
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AHA2 orientation in liposomes was determined by site-specific labeling of the SNAP-tag. Proteoliposomes (10 µL, ~500 ng AHA2) were incubated for 2 h either sequentially, first with 20 pmol membrane impermeable SNAP dye (10 µM SNAP-Surface® 488, New England BioLabs Inc., Ipswich, MA, USA) followed by 20 pmol membrane permeable SNAP dye (10 µM SNAP-Cell® 647-SiR, New England BioLabs Inc.) in reconstitution buffer supplemented with 1 mM DTT or with each dye separately. Samples were analyzed by SDS-PAGE using 12% gels and visualized on a ChemiDoc XRS Imaging System (Bio-Rad Laboratories GmbH, München, Germany) using the Image Lab™ software (https://www.bio-rad.com/zh-cn/product/image-lab-software?ID=KRE6P5E8Z&WT_mc_id=211202033049&WT_srch=1&WT_knsh_id=cr180107&gclid=EAIaIQobChMI0eKynPLxgAMVlZhmAh2HUArSEAAYASABEgJPdvD_BwE ) and pre-programmed option for Coomassie stained gels or Alexa488/Alexa647 fluorophores, respectively [55 (link)].
6
Fluorescent Labeling of SNAP-Tagged Proteins
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Labelling of SNAP-YuaG or free SNAP for microscopy was performed as described before [17 (link)]. Briefly, for labelling with the cell permeable SNAP-Cell TMR-Star (NEB) (excitation maxima: 554 nm; emission maxima: 580 nm) or the cell impermeable SNAP-Surface 488 (NEB) (excitation maxima: 506 nm; emission maxima: 526 nm) 1 µl of the stock solution was added to 400 µl culture and incubated for 30 minutes at 37°C. Cells were washed twice with fresh CH media,incubated for 15 minutes at 37°C and analysed microscopically.
Microscopy was performed as described in Bach et al. 2014 [32 (link)]. Images were taken on Zeiss AxioImager M1 equipped with a Zeiss AxioCam HRm camera. An EC Plan-Neofluar 100x/1.30 Oil Ph3 objective was used. Digital images were acquired with the AxioVision (Zeiss) software and analysed using the AxioVision 4.6 software (Zeiss). Final image preparation was done in Adobe Photoshop CS2 (Adobe Systems Incorporated).
Microscopy was performed as described in Bach et al. 2014 [32 (link)]. Images were taken on Zeiss AxioImager M1 equipped with a Zeiss AxioCam HRm camera. An EC Plan-Neofluar 100x/1.30 Oil Ph3 objective was used. Digital images were acquired with the AxioVision (Zeiss) software and analysed using the AxioVision 4.6 software (Zeiss). Final image preparation was done in Adobe Photoshop CS2 (Adobe Systems Incorporated).
7
Notch1, EGFR, and β-Catenin Signaling Assays
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Antibodies against Notch1 ICD (D1E11, 1:100 IF, 1:1,000 WB), Notch1 V1744 (D3B8, 1:500 WB), EGFR (D38B1, 1:100 IF, 1:1,000 WB), pEGFR (D7A5, 1:1,000 WB), GFP (D5.1, 1:1,000 WB), non-phospho (active) β-catenin (D13A1, 1:1,000 WB), GAPDH (14C10, 1:10,000 WB), and YAP (D8H1X, 1:200 IF) were from Cell Signaling Technologies. β-catenin antibody (14, 1:1,000 WB) was from BD Biosciences. E-cadherin antibody (HECD-1, 1:1,000 IF, 1:1,000 WB) was from Takara Bio. Notch1 ECD (ABS90, 1:1,000) was from Millipore. FAM83H antibody (1:1,000 WB) was from Bethyl Laboratories. Lamin B1 antibody (12987-1-AP, 1:1,000) was from Proteintech. TexasRed-EGF, rhodamine phalloidin, and Alexa Fluor 488, 568, and 647 goat anti-mouse and anti-rabbit IgG secondary antibodies (1:400) were from Invitrogen. Alexa Fluor 647 azide and EdU cell proliferation kit were from Invitrogen. Hoescht and DAPT were from Sigma. Anti-SNAP (P9310S, 1:1,000), SNAP-Capture, and SNAP-Surface 488 were from New England Biolabs.
8
Visualizing Protein Tags in Brain Slices
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SNAP, CLIP, and HaloTags were visualized with their substrates, SNAP-Surface 488 (#S9124S, New England Biolabs), HaloTag TMR Ligand (#G8252, Promega), CLIP-Surface 647 (#S9234S, New England Biolabs), respectively. Brain slices of 220 or 1000 μm thickness were incubated with the substrates (2 μM each) in 2 ml 2% saponin in PBS overnight. The slices were then washed with PBS (3 × 30 min).
9
Cell Surface Protein Labeling and Crosslinking
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We obtained all BG- and BC-functionalized derivatives, including the bifunctional cross-linkers (synthesis see above), CLIP-Surface488 and SNAP-Surface488 from New England Biolabs (NEB) and used them according to the manufacturer's instructions. Briefly, we prepared a stock of 1 mM tag in DMSO and labeled cells expressing cell-surface CLIP- or SNAP-fusion proteins by incubating them in 5 μM tag in full media for 30 min, followed by three washes. We performed all imaging of PFA fixed, surface labeled cells using a 63× objective on an LSM710 confocal microscope (Zeiss). 3D reconstructions of the images are shown to illustrate surface staining.
We slightly altered the above protocol for crosslinking cultured hippocampal neurons. Briefly, we incubated the cells with 2.5 μM bifunctional crosslinker in complete neuron media for 30 min. We then blocked all unreacted SNAP and CLIP epitopes by incubating the cells in 10 μM each of SNAP-cell block and CLIP-cell block (NEB S9106S and S9220S) in full neuron media for 30 min. We then washed the cells three times with full neuron media and finally in PBS.
We slightly altered the above protocol for crosslinking cultured hippocampal neurons. Briefly, we incubated the cells with 2.5 μM bifunctional crosslinker in complete neuron media for 30 min. We then blocked all unreacted SNAP and CLIP epitopes by incubating the cells in 10 μM each of SNAP-cell block and CLIP-cell block (NEB S9106S and S9220S) in full neuron media for 30 min. We then washed the cells three times with full neuron media and finally in PBS.
10
Visualizing SNAP-FZD5 Receptor Internalization
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HCT116 cells were grown on 8-well Lab-Tek II chamber sides (Thermo Scientific). After 48 h cells were co-transfected with 1μg of SNAP-FZD5 (kindly given by Dr. Mariann Bienz)
[2] and 1 μg of C-terminal FLAG-RNF43 plasmids. Cells were labelled with 1 μM SNAP-surface488 (New England Biolabs) substrate at room temperature for 15 min. After labeling medium was removed, cells were washed with growth medium once, then new growth media was added. Labelled surface FZD5 proteins were chased for 5 or 30 min at 37 °C, then cells were fixed with 4% PBS-buffered formaldehyde solution at room temperature for 10 min.
After PBS washing 3 times, cells were permeabilized with 0.2% Triton X-100 in PBS for 10
[2] and 1 μg of C-terminal FLAG-RNF43 plasmids. Cells were labelled with 1 μM SNAP-surface488 (New England Biolabs) substrate at room temperature for 15 min. After labeling medium was removed, cells were washed with growth medium once, then new growth media was added. Labelled surface FZD5 proteins were chased for 5 or 30 min at 37 °C, then cells were fixed with 4% PBS-buffered formaldehyde solution at room temperature for 10 min.
After PBS washing 3 times, cells were permeabilized with 0.2% Triton X-100 in PBS for 10
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