1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L -α-phosphatidylinositol (liver PI), DOPS, PIP2, BSM, di-stearoyl phosphatidyl ethanolamine-PEG(2000)-biotin and cholesterol were purchased from Avanti Polar Lipids. Fluorescent lipid bodipy-tetra-methyl-rhodamine-PIP2 (TMR-PIP2) were purchased from Echelon Biosciences. Two lipid compositions were used—(1) 25% DOPC+30% DOPE+5% liver PI+30% DOPS+10% PIP2 supplemented with 15% cholesterol and 1% TMR-PIP2; (2) 60% BSM+30% DOPS+10% PIP2+1% TMR-PIP2 supplemented with 50% cholesterol. streptavidin-coated polystyrene beads (3.05 μm) used for nanotube pulling experiments were purchased from Spherotech. Clathrin was extracted from fresh porcine brains and purified as described in ref. 15 (link). Clathrin was labelled with Alexa Fluor 488 C5 maleimide (Invitrogen). AP180 was expressed in BL21/DH3α cells and purified by glutathione S transferase pull-down and glutathione S transferase cleaved by PreScission protease. AP180 was labelled with Alexa Fluor 488 C5 maleimide (Invitrogen). Epsin was purified from cells infected with baculovirus as described in ref. 5 (link) and labelled with Alexa Fluor 488 C5 maleimide (Invitrogen). All proteins were aliquoted in GTPase buffer (HEPES 20 mM pH=7.4, NaCl 100 mM and MgCl2 1 mM ) and stored at −80 °C.
Alexa fluor 488 c5 maleimide
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Australia
Alexa Fluor 488 C5 maleimide is a fluorescent dye used for labeling proteins and other biomolecules. It is a reactive dye that forms a covalent bond with thiol groups, allowing for the specific labeling of cysteine residues within proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 647 c2 maleimide, by Thermo Fisher Scientific (7 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (7 mentions)
Pd 10 desalting column, by GE Healthcare (4 mentions)
Thioflavin t, by Merck Group (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 c5 maleimide, by Thermo Fisher Scientific (3 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (3 mentions)
Sephadex g 25, by GE Healthcare (3 mentions)
Bradford protein assay kit, by Bio-Rad (3 mentions)
Arvo mx 1420, by PerkinElmer (3 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Ni2 affinity chromatography, by Qiagen (2 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (2 mentions)
Nanophotometer, by Implen (2 mentions)
Alexa fluor 568 c5 maleimide, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (2 mentions)
Badan, by Thermo Fisher Scientific (2 mentions)
Mo control software, by NanoTemper (2 mentions)
Cy3 maleimide, by GE Healthcare (2 mentions)
118 protocols using alexa fluor 488 c5 maleimide
1
Lipid Composition and Protein Labeling
2
Alexa Fluor 488 Labeling of Galectin-3
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Human galectins were prepared as outlined previously. The recombinant galectin-3 was purified by affinity chromatography on lactosyl-Sepharose, and the bound lectin was eluted with 100mM lactose in PBS and 14mM β-mercaptoethanol (β-ME). Prior to derivatization, β-ME was removed from the galectin-3 samples by passing through a PD-10 gel filtration column (GE Healthcare), followed by the addition of 100mM lactose to help maintain the stability of galectin-3 and reduce the likelihood of adduct formation at or near the carbohydrate recognition domain. Alexa Fluor 488-labeled galectin-3 was prepared with either Alexa Fluor 488 C5-maleimide or Alexa Fluor 488 carboxylic acid succinimidyl ester dilithium salt-reactive dyes (Molecular Probes) according to manufacturer’s protocol.
3
Cys-Z2891 Protein Labeling with Alexa Fluor 488
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Cys-Z2891 (700 µg) was diluted to 100 nM and reduced with 20 mM dithiothreitol (DTT) for 45 min at 37°C. DTT was removed in NAP-5 columns equilibrated in PBS, and 500 nmol (5X molar excess) Alexa Fluor® 488 C5-maleimide (Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA) dissolved in DMSO was added. After incubation at 4°C overnight, unbound Alexa488 was removed in a PD-10 column (GE Healthcare Life Sciences) equilibrated with PBS. Degree of labelling and protein concentration were determined using a NanoDrop ND-1000 (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
4
Cysteine Labeling of Native Nucleosomes
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200 µL of native nucleosome were gently mixed with 250 µL of buffer (10 mM Tris-HCl, 0.7 mM EDTA, 3.6 M NaCl). 5-fold molar excess of AlexaFluor488 C5-maleimide (Molecular Probes) was added and the mixture was incubated for 3 hours in the dark, at room temperature. After that, successive dialyses against solutions of decreasing ionic force was carried out following a modified protocol from Oudet P. et al.33 (link). The solution was dialysed (3.5 kDa MWCO) in the dark at +4 °C against 500 mL of solution containing 10 mM Tris-HCl, 0.7 mM EDTA and decreasing NaCl concentration: 2 M for 2 hours, 1.5 M for 12 hours, 1 M for 24 hours, 0.75 M for 12 hours, 0.5 M for 24 hours, 0.4 M for 12 hours and a last dialysis with a new MWCO (20 kDa MWCO) against 0.4 M of NaCl for 12 hours. The DNA concentration was measured (dilution 1:100 in 0.1% (v/v) SDS solution). The samples were stored at −20 °C and referred as “cysteine nucleosome”.
5
Electrophysiological Characterization of KCNQ1 Channels
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Microelectrodes were made with thin wall borosilicate glass (B150-117-10, Sutter Instrument, Novato, CA) by a micropipette puller (P-97 or P-1000, Sutter Instrument, Novato, CA). The pipette resistance was 0.5 to 3 megohm when filled with 3 M KCl solution and submerged in ND96 solution. TEVC and VCF experiments were recorded in ND96 solutions at room temperature. Whole-cell currents were recorded with a CA-1B amplifier (Dagan, Minneapolis, MN) driven by Patchmaster (HEKA, Holliston, MA) software. Current recordings were sampled at 1 kHz and low-pass–filtered at 2 kHz. For VCF experiments, the KCNQ1 channels were labeled by incubating oocytes expressing KCNQ1 channels for 1 hour on ice in 10 μM Alexa Fluor 488 C5-maleimide (Molecular Probes, Eugene, OR) in high K+ solution [98 mM KCl, 1.8 mM CaCl2, and 5 mM Hepes (pH 7.6)]. The oocytes were washed and incubated in ND96 after labeling. VCF was performed by recording currents with the same instruments as TEVC. The fluorescence signals were measured by a Pin20A photodiode (OSI Optoelectronics, Hawthorne, CA) and an EPC10 patch clamp amplifier (HEKA, Holliston, MA) sampled at 1 kHz and filtered at 200 Hz. The EPC10 amplifier was controlled by the CA-1B amplifier to ensure simultaneous current and fluorescence measurements. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
6
Fluorescent Labeling of Bacterial Structures
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Fluorescence microscopy was performed using a Zeiss Axio Imager A1 equipped with an EVOLVE EMCCD camera (Photometrics) and a TIRF objective with an aperture of 1.45. Picture acquisition was done using VisiView (2.1.2). Time-lapse microscopy was performed using a 515 nm laser illumination. Super resolution microscopy was done using Leica G-STED SP8 microscope. Images were acquired with three or four line scans at 200 Hz. Further analysis was done with Metamorph 6.5 (MDS Analytical Technologies) and ImageJ (National Institutes of Health, Bethesda, MD), MTrackJ (ImageJ plugin) was used to generate the tracks.
For fluorescent microscopy of the cysteine-labeled hook and flagella, cells were pelleted (0.5 ml of LB broth at 0.5–2.0 OD600) by delicate centrifugation at 3500–5000 rpm for 10 min to avoid the shearing of the flagella or hook, and washed once with 1 ml T-Base buffer [15 mM (NH4)2SO4, 80 mM K2HPO4, 44 mM KH2PO4, 3.4 mM sodium citrate, and 3.0 mM MgSO4·6H20]. Cells were resuspended in 50 μl of T-Base buffer containing 5 μg/ml Alexa Fluor 488 C5 maleimide (Molecular Probes), and incubated for 5 min at room temperature. Cells were then washed once with 500 μl T-Base buffer.
For fluorescent microscopy of the cysteine-labeled hook and flagella, cells were pelleted (0.5 ml of LB broth at 0.5–2.0 OD600) by delicate centrifugation at 3500–5000 rpm for 10 min to avoid the shearing of the flagella or hook, and washed once with 1 ml T-Base buffer [15 mM (NH4)2SO4, 80 mM K2HPO4, 44 mM KH2PO4, 3.4 mM sodium citrate, and 3.0 mM MgSO4·6H20]. Cells were resuspended in 50 μl of T-Base buffer containing 5 μg/ml Alexa Fluor 488 C5 maleimide (Molecular Probes), and incubated for 5 min at room temperature. Cells were then washed once with 500 μl T-Base buffer.
7
Fluorescent Labeling of Bacterial Flagella
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Fluorescence microscopy was performed with a Nikon 80i microscope with a phase contrast objective Nikon Plan Apo 100X and an Excite 120 metal halide lamp. FM4–64 was visualized with a C-FL HYQ Texas Red Filter Cube (excitation filter 532–587 nm, barrier filter >590 nm).
For fluorescent microscopy of flagella, 0.5 ml of broth culture was harvested at 0.5–2.0 OD600, and washed once in 1.0 ml of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4). The suspension was pelleted, resuspended in 50 µl of PBS buffer containing 5 µg/ml Alexa Fluor 488 C5 maleimide (Molecular Probes), and incubated for 5 min at room temperature50 (link). Cells were then washed twice with 500 µl PBS buffer. When appropriate, membranes were stained by resuspension in 50 µl of PBS buffer containing 5 µg/ml FM4–64 (Molecular Probes) and incubated for 10 min at room temperature. Three microliters of suspension were placed on a microscope slide and immobilized with a poly-L-lysine-treated coverslip.
For fluorescent microscopy of flagella, 0.5 ml of broth culture was harvested at 0.5–2.0 OD600, and washed once in 1.0 ml of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4). The suspension was pelleted, resuspended in 50 µl of PBS buffer containing 5 µg/ml Alexa Fluor 488 C5 maleimide (Molecular Probes), and incubated for 5 min at room temperature50 (link). Cells were then washed twice with 500 µl PBS buffer. When appropriate, membranes were stained by resuspension in 50 µl of PBS buffer containing 5 µg/ml FM4–64 (Molecular Probes) and incubated for 10 min at room temperature. Three microliters of suspension were placed on a microscope slide and immobilized with a poly-L-lysine-treated coverslip.
8
Fluorescent Probes for Molecular Imaging
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Alexa
Fluor 488 C5 maleimide, Alexa Fluor
647 C2 maleimide, Alexa Fluor 405 NHS ester, and Alexa Fluor 647 NHS
ester were purchased from Molecular Probes. Heparin (low molecular
weight Heparin) was obtained from Fisher Scientific UK. Ammonium acetate,
thioflavin T (ThT), and dithiothreitol (DTT) were purchased from Sigma.
pFTAA was a kind gift from Therese Klingstedt.
Fluor 488 C5 maleimide, Alexa Fluor
647 C2 maleimide, Alexa Fluor 405 NHS ester, and Alexa Fluor 647 NHS
ester were purchased from Molecular Probes. Heparin (low molecular
weight Heparin) was obtained from Fisher Scientific UK. Ammonium acetate,
thioflavin T (ThT), and dithiothreitol (DTT) were purchased from Sigma.
pFTAA was a kind gift from Therese Klingstedt.
9
Fluorescent Protein Incorporation via Amber Codon
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The DsRed expression vector including an amber codon at codon position 131 (pcDNA5-DsRed-131amber) was prepared by subcloning the DsRed gene from pDsRed-Express-N1 (Clontech) into pcDNA5/FRT/TO Flp-In expression vector (Invitrogen) between BamHI and XhoI sites, and then mutating the codon position 131 by using a QuikChange site-directed mutagenesis kit (Stratagene). CHO cells stably expressing DsRed mRNA-131amber (CHO-DsRed-131amber) were prepared by transfecting the Flp-In CHO cell line (Invitrogen) with pcDNA5-DsRed-131amber according to the standard protocol of the Flp-In system. The CHO-DsRed-131amber cells were grown at 37 °C and 5% CO2 in Ham's F12 medium supplemented with 10% fetal bovine serum (Sigma) and 1% streptomycin/penicillin (Gibco).
CHO-DsRed-131amber cells plated in a 35 mm dish (∼70% confluence, in Ham's F12 without antibiotics) were microinjected with 1 × PBS solution including 36 μM DEACM-PPG-tRNACUA and, as an injection marker, 10 μM Alexa Fluor 488 C5 maleimide (Molecular Probes). Microinjections were performed using Eppendorf Femtojet microinjection equipment and Femtotip microinjection capillary tips at 100–150 hPa. The cells were irradiated for 30 s by using the Hg–Xe lamp (∼405 nm, 125 mW cm−2) and then incubated for 4 h at 37 °C and 5% CO2. Cellular fluorescence was imaged using a fluorescence microscope (Olympus IX51/IX2-FL-1/MP5Mc/OL-2).
CHO-DsRed-131amber cells plated in a 35 mm dish (∼70% confluence, in Ham's F12 without antibiotics) were microinjected with 1 × PBS solution including 36 μM DEACM-PPG-tRNACUA and, as an injection marker, 10 μM Alexa Fluor 488 C5 maleimide (Molecular Probes). Microinjections were performed using Eppendorf Femtojet microinjection equipment and Femtotip microinjection capillary tips at 100–150 hPa. The cells were irradiated for 30 s by using the Hg–Xe lamp (∼405 nm, 125 mW cm−2) and then incubated for 4 h at 37 °C and 5% CO2. Cellular fluorescence was imaged using a fluorescence microscope (Olympus IX51/IX2-FL-1/MP5Mc/OL-2).
10
Quantifying Plasma Membrane Thiols
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For detection of external plasma membrane thiols, cells were seeded in precoated 12-well tissue culture plates and grown at 37 °C in 5% CO2. After 48 hours of incubation, monolayers were incubated for 2 hours in antibiotic-free medium containing 2% FBS. After 3 hours of incubation with or without 1 mM DTT or 1 mM GSH or after 1 hour with or without 1 mM DTNB, the monolayers were washed and incubated in PBS alone or with 10 μM Alexa fluor 488 C5-maleimide (Molecular Probes) for 30 minutes at 37 °C. Subsequently, the cells were washed and scraped in PBS. Labeled thiols were detected by cytofluorimetric analysis using a BD FACScalibur.
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