VAMP2-SEP and synaptotagmin1-SEP were kind gifts from Jürgen Klingauf. GCaMP6f was obtained from Addgene (#40755). VaChT-SNAP and VaChT-pHuji were created by replacing the open reading frame of SEP for either SNAP-tag (New England Biolabs) or pHuji5 (link) in the VAChT-SEP construct15 (link). VGluT1-SNAP and VGluT1-pHuji were created by replacing the open reading frame of SEP for either SNAP-tag (New England Biolabs) or pHuji5 (link) in the VGluT1-SEP construct17 (link). VAMP2-pHuji was created by swapping the SEP gene for pHuji in VAMP2-SEP sensor construct2 (link).
Snap tag
Manufactured by New England Biolabs
Sourced in United States
The SNAP-tag is a protein fusion tag that can be used to covalently label proteins with a wide variety of synthetic molecules. It is based on a human DNA repair enzyme that can react specifically and efficiently with benzylguanine or benzylchloropyrimidine derivatives. The SNAP-tag can be fused to a target protein, expressed in cells, and subsequently labeled with a synthetic probe molecule.
Automatically generated - may contain errors
Lab products found in correlation
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Pshuttle cmv vector, by Agilent Technologies (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
P chk1, by Cell Signaling Technology (2 mentions)
Hsp70, by BD (2 mentions)
Sc 376450, by Santa Cruz Biotechnology (2 mentions)
Myc tag, by Cell Signaling Technology (2 mentions)
γ tubulin, by Merck Group (2 mentions)
Plvx ires puro, by Takara Bio (1 mentions)
Hepes and sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Annexin 5, by Santa Cruz Biotechnology (1 mentions)
Dusp2, by Santa Cruz Biotechnology (1 mentions)
P44 42 mapk, by Cell Signaling Technology (1 mentions)
Nap 5 column, by GE Healthcare (1 mentions)
Halotag7, by Promega (1 mentions)
Dna sequencing, by Eurofins (1 mentions)
Gcamp6f, by Addgene (1 mentions)
Egfp actin, by Takara Bio (1 mentions)
18 protocols using snap tag
1
Fluorescent Protein Fusion Constructs
2
Construction of Fluorescently-Tagged GPCR and Effector Proteins
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SSF-DOR construct consists of an N-terminal signal sequence followed by a Flag tag followed by the mouse DOR sequence in a pcDNA3.1 vector backbone. To create SNAP-DOR, the full-length receptor sequence was amplified from the SSF-DOR construct by PCR with compatible cut sites (BamHI and XbaI). The SNAP tag (New England Biolabs, Ipswich, MA) was amplified by PCR with compatible cut sites (HindIII and BamHI) and both were ligated into a pcDNA3.1 vector backbone to produce the final construct containing an N-terminal signal sequence, followed by the SNAP tag and then the receptor. βarrestin-1 was generated from a geneblock (Integrated DNA Technologies, Coralville, IA) containing the human cDNA (ENST00000420843) for hARRB1 with HindIII and AgeI cut sites. mScarlet was amplified by PCR from pmScarlet_alphaTubulin_C1 a gift from Dorus Gadella (Addgene plasmid #85045) (74) , with AgeI and XbaI cut sites. Both were then ligated into a pcDNA3.1 vector backbone to produce a Cterminally tagged β-arrestin-1. β-arrestin 2 tagged with tdTomato was generated from β-arrestin 2-GFP via restriction site cloning (44) . Nb39-mVenus was a gift from Drs. Bryan Roth and Tao Che. Venus-miniGsi and Venus-miniGs were gifts from Drs. Greg Tall and Nevin Lambert. pcDNA3-ICUE3 was a gift from Dr. Jin Zhang (Addgene plasmid #61622) (46) .
3
Immortalized Mouse Embryonic Fibroblasts
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The vim+/+ and vim−/− mEFs were derived from vim+/+ and vimentin-null mice and immortalized by stable expression of SV40 large T antigen (kindly provided by J. Eriksson, Abo Akademi University, Turku, Finland). Cells were cultured in DMEM with 25 mM Hepes and sodium pyruvate (Life Technologies) supplemented with 10% FBS, 1% penicillin/streptomycin, and nonessential amino acids. All cell cultures were maintained at 37°C and 5% CO2.For the reconstitution of vimentin protein expression in vim−/− mEFs, the vimentin cDNA was expressed from pLVX-IRES-neo (Clonetech) by lentiviral transduction. To express the SNAP-vimentin fusion protein, the vimentin cDNA was subcloned into pLVX-IRES-puro (Clontech) with the addition of a SNAP tag (NEB). After selection of the transduced cells with the appropriate antibiotic for 1 wk vimentin expression was assessed by Western blotting using mouse anti-vimentin antibody (Sigma, clone V9, V6630) as described before (Gan et al., 2016 (link)).
4
Fluorescent Protein Labeling Protocol
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Cells expressing the SNAP-tag (New England Biolabs) and/or HaloTag (Promega) fusion protein were labeled following the manufacturer’s preparation protocol (www.neb.com and www.promega.co.uk ). The cell medium was first replaced with 200 μl of SNAP-tag (or HaloTag) medium [Leibovitz’s L-15 medium (Life Technologies) with 1 to 5 μM SNAP-tag (or HaloTag) ligand] and then incubated for 45 min at 37°C at 300 rpm. Finally, to ensure that free dye would not remain in the cytoplasm, the cell-labeling solution was washed three times in L-15 and then further incubated for 30 min at 37°C at 300 rpm.
5
Comprehensive Protein Expression Analysis
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Whole cell lysates were collected by RIPA buffer and subjected to Western blotting as described previously [18 (link)]. Antibodies against VEGF-C (GeneTex, GTX113574), DUSP2 (Santa Cruz, sc-32776), Phospho-p44/42 MAPK (Cell Signaling Technology #4370), p44/42 MAPK (Cell Signaling Technology #4696), CD63 (Taiclone, tcba9211), GAPDH (Genetex GTX100118), Furin (Abcam, ab183495), PC5/6 (Abcam, ab40133), PCSK7 (Abcam, ab47925), HSP70 (Santa Cruz, sc-32239), Annexin V (Santa Cruz, sc-74438), SNAP-tag (New England Biolabs, P9310 S) were used for Western blotting.
6
Engineered β1-adrenoceptor Fusion Constructs
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The β1-yellow fluorescent protein (YFP)N and β1-YFPC receptor constructs were generated by fusing either the N-terminal fragment of YFP (YFPN; amino acids 1–155) or the C-terminal fragment of YFP (YFPC; amino acids 156–239) to the C-terminal end of the full-length wild-type human β1-adrenoceptor. The SNAP-β1 construct was generated by fusing the SNAP-tag (New England Biolabs, Ipswich, MA, USA) to the N-terminal end of the wild-type human β1-adrenoceptor. The D138A mutation (7 (link)) was introduced into the β1-YFPC and the SNAP-β1 sequence using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Cheshire, United Kingdom). All sequences were confirmed by DNA sequencing. All receptor constructs were subcloned into pcDNA3.1 vectors.
7
Fluorescent Labeling of Kinesin Constructs
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To produce dimeric kinesin constructs HK432 and KIF21A-552, amino-acid residue 1–432 of KIF5B and residue 1–552 of KIF21A were each fused to a spacer (GGGGSGGGGS), a SNAP tag (New England Biolabs), a spacer (G) and a His6 tag, respectively. For the monomeric kinesin construct HK349, amino-acid residues 1–349 of KIF5B were fused to a spacer (GGGGSGGGGS), SNAP tag, a spacer residue (G) and a His6 tag (K349-SNAP-His). These constructs were expressed in E. coli then purified using immobilized metal-affinity chromatography (TALON, Clontech). The purified proteins were labelled with fluorescent dye BG-549 (SNAP-surface 549; New England Biolabs) at a dye-to-protein ratio of 2:1–5:1 for 60 min at RT, and separated from the free dye by gel filtration using a NAP-5 column (GE Healthcare) and by MT-affinity purification. The stoichiometry of the labelling was >0.9, as determined from the concentrations of both BG-549 and kinesin protein.
8
Engineering Myosin Constructs for Structural Studies
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For wild type subfragment-1 (S1) of the myosin construct, human skeletal muscle myosin IIa cDNA (Kazusa Product ID FXC25901, Kisarazu, Japan) was truncated at Ala849. This fragment included the motor domain, essential light chains (ELC) binding domain and regulatory light chains (RLC) binding domain. For oligonucleotide labeling and protein purification, SNAP-tag (New England Biolabs Inc.), FLAG-tag, and 6 × His-tag were attached at the C-terminal via linkers (3 a.a., GGL). Two amino acids (Leu-Glu) corresponding to the restriction endonuclease recognition site (XhoI: CTCGAG) were kept between SNAP-tag and FLAG-tag. For the light chain null construct (lever-arm-less myosin S1), ELC, and RLC binding sites (Lys786-Leu846) were deleted from the S1 construct. These myosin fragments were introduced downstream of the multi cloning site of the pShuttle-CMV vector (Agilent Technologies, Santa Clara, CA, USA). For the double lever-arm length construct (double lever-arm length Myosin S1), the ELC and RLC binding domains were inserted just after Ala849 of WT S1.
9
Immunoblotting Antibodies for DNA Repair
10
Immunoblotting Antibodies for DNA Repair
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Immunoblotting was performed as described19 (link) with the following antibodies: 53BP1 (175933, Abcam; 1:1000; 100-304, Novus Biological; 1:1000); BRCA1 (MAB22101, R+D systems; 1:500); BRCA2 (OP95, Millipore; 1:500); CHK1 (8408, Santa Cruz; 1:1000); pCHK1 (2341, Cell Signaling Technology; 1:1000); CHK2 (611570, BD; 1:1000); Flag-tag (M2, Sigma; 1:1000); γ-Tubulin (GTU488, Sigma; 1:20,000); GFP (11814460001, Sigma; 1:1000); HA (3724, Cell Signaling Technology; 1:20,000); HSP70 (610608, BD; 1:1000); MAD2L2/REV7 (180579, Abcam; 612266, BD; 1:1000); Myc-tag (9B11, Cell Signaling Technology; 1:1000); OBFC1/STN1 (89250, Abcam; 1:1000; SC-376450, Santa Cruz; 1:1000); PRIM1 (10773-1-AP, Proteintech; 1:1000); RIF1 (#1240, de Lange Lab; 1:1000); SHLD1 (PA5-59280, Thermo-Fisher; 1:1000); SNAP tag (9310, NEB; 1:1000); TRF2 (#1254, de Lange Lab; 1:5,000). Affinity-purified peptide antibodies against mouse SHLD1 and SHLD2 proteins (Chapman Lab, unpublished; 1:1000) were generated by Eurogentec.
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