Pd spintrap g 25

Manufactured by Cytiva

Sourced in Japan

The PD SpinTrap™ G-25 is a laboratory device designed for the rapid desalting and buffer exchange of small sample volumes. It utilizes a size-exclusion chromatography principle to separate target molecules from unwanted salts, buffers, and other small molecules.

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Lab products found in correlation

Superdex 200 increase 10 300 gl, by Cytiva (1 mentions) 96 well black bottom plates, by Corning (1 mentions) Gen5 3, by Agilent Technologies (1 mentions) Prism 9, by GraphPad (1 mentions) Hilyte647, by AnaSpec (1 mentions) P96 1.5h n, by Cellvis (1 mentions) Sp8 confocal microscope, by Leica (1 mentions) Pti quantamaster 400, by Horiba (1 mentions) Pierce detergent removal spin column, by Thermo Fisher Scientific (1 mentions)

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PD SpinTrap G-25 column

8 protocols using pd spintrap g 25

Labeling of Antibody IP3G2 with Dy-654

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Of the antibody stock solution (IP3G2), containing 3.75 mg mL⁻¹ IP3G2 and 0.1 wt.% NaN₃ in PBS, 53.3 µL (1.33 nM) were diluted with 46.7 µL of PBS-C (100 mM phosphate and 137 mM sodium chloride pH 7.8) to a final volume of 100 µL and a concentration of approx. 2 g L⁻¹. A PD SpinTrap G-25 (Cytiva) was conditioned four times with 140 µL of PBS-C at 800 g and 4 °C. The diluted IP3G2 solution (100 µL) was transferred to the conditioned SpinTrap, and an additional stacking buffer of 40 µL PBS-C was added. The SpinTrap was centrifuged at 800 g for one minute at 4 °C, and the eluate, 140 µL, was collected.
1.68 µL (7.6 nM) of 4.5 mM Dy-654-NHS in DMF, prepared as described in [46 (link)], were added to the eluate, a six-fold molar excess, and shaken for 2 h at 800 rpm and 21 °C in the dark and subsequently stored for 96 h at 4 °C in the fridge. The conjugate was purified by size exclusion chromatography (SEC) with a PD SpinTrap G-25 (Cytiva) and conditioned with PBS as described above. To the 140 µL eluate, containing approx. 1.25 mg mL⁻¹ IP3G2-Dy-654, 5 vol.% of 1:100 diluted ProClin300 was added as a preservative. The labeled antibody was stored in the dark at 4 °C until further use and remained stable for several months.

Paul M., Tannenberg R., Tscheuschner G., Ponader M, & Weller M.G. (2021). Cocaine Detection by a Laser-Induced Immunofluorometric Biosensor. Biosensors, 11(9), 313.

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Sulfide Production from L-Cysteine

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To produce free sulfide from L‐Cys, 10 μM of E. coli desulfurase IscS was incubated at 25°C overnight with 1 mM L‐Cys and 20 μM PLP cofactor in a desulfurase buffer (20 mM Hepes pH 8.5, 150 mM NaCl, 2 mM MgCl₂). To examine the fate of sulfur in the yeast system, 10 μM of ScUba4 and 40 μM of ScUrm1 were added, with 2.5 mM ATP and 1 mM TCEP, and incubated for 1 h at 30°C. Analogously, for Ct., 10 μM of CtUba4 and 40 μM of CtUrm1_C55S were added with 2.5 mM ATP and 1 mM TCEP, and incubated for 1 h at 37°C. Following incubation, the samples were desalted using PD SpinTrapTM G‐25 (Cytiva) columns, buffer exchanged into 40 mM ammonium acetate, and analyzed by ESI‐MS as previously described.

Ravichandran K.E., Kaduhr L., Skupien‐Rabian B., Shvetsova E., Sokołowski M., Krutyhołowa R., Kwasna D., Brachmann C., Lin S., Guzman Perez S., Wilk P., Kösters M., Grudnik P., Jankowska U., Leidel S.A., Schaffrath R, & Glatt S. (2022). E2/E3‐independent ubiquitin‐like protein conjugation by Urm1 is directly coupled to cysteine persulfidation. The EMBO Journal, 41(20), e111318.

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Preparation and Characterization of Thiocarboxylated Urm1

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Uba4‐mediated thiocarboxylation of Urm1 was performed as described (Termathe & Leidel, 2018 (link)). Briefly, 20 μM of Urm1 was mixed with 10 μM of Uba4 in the thiocarboxylation buffer (20 mM HEPES pH 8.5, 150 mM NaCl, and 2 mM MgCl₂), supplemented with 5 mM ATP, 5 mM TCEP and 180 μM of sodium thiosulfate (Na₂S₂O₃) was added to the reaction mix and incubated for 1 h at 30°C. For Urm1‐SH, samples were desalted using PD SpinTrapTM G‐25 (Cytiva) columns and either loaded on SDS–PAGE gels supplemented with 20 μM of APM to visualize the shift of the thiol group or buffer exchanged into 40 mM ammonium acetate and analyzed by ESI‐MS. Thiocarboxylation of Urm1 was scaled up and Urm1‐SH was purified by using Superdex 200 Increase 10/300 GL (Cytiva) on ÄKTA™ start system. The purified Urm1‐SH was snap frozen and stored at −80°C in storage buffer (20 mM Tris pH 7.5 and 200 mM NaCl) for further use.

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Kinetic analysis of LpxC inhibitors

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Inhibitor residence times were determined by monitoring the rate of association of a fluorescent probe to the enzyme-inhibitor complexes using a Penefsky column-based method. Fifty μL of a solution containing 10 μM purified ecLpxC and 20 μM of LpxC inhibitor (CHIR-090, PT810 or PT805) in 25 mM NaH₂PO₄, pH 8.0 buffer containing 300 mM KCl and 0.1 mg/mL BSA, was incubated at 25 °C for 16 h. Then, 5 μL of the incubated mixture was rapidly diluted into 1 mL of the reaction buffer containing 1 μM fluorescence competitor PT900 at 25 °C. Subsequently, 100 μL aliquots of the diluted mixture were collected at different time points and loaded onto the spin-column (PD SpinTrap™ G-25, Cytiva), which was then centrifuged in a swinging bucket rotor (Eppendorf 5810R, 15-amp version) at 800xg for 2 min. One hundred μL aliquots of the eluate were dispensed into 96 well black bottom plates (Corning, NY) in duplicate, and the fluorescence intensity was quantified using a plate reader (BioTek, Gen5, 3.09) at λ_ex 315 nm and λ_em 420 nm. The change in fluorescence as a function of time was fit to a one-phase association equation in GraphPad Prism 9.0. The rate of PT900 association to ecLpxC was taken to be equivalent to the overall off rate for dissociation of the inhibitor from ecLpxC (k_off), and residence times (t_R) were calculated by taking the reciprocal of k_off (t_R = 1/k_off).

Basu R., Wang N., Basak S., Daryaee F., Babar M., Allen E.K., Walker S.G., Haley J.D, & Tonge P.J. (2021). The impact of target turn-over on the translation of drug-target residence time to time-dependent antibacterial activity. ACS infectious diseases, 7(9), 2755-2763.

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Fluorescence Microscopy of Protein Labeling

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Fluorescence microscopic images of the reaction with labeled proteins were obtained using Leica SP8 confocal microscope at 40x magnification in a clear glass bottom 96 well black plates (P96-1.5H-N, Cellvis Inc.). Briefly, protein labeling was carried out by incubating three molar excess of fluorescent dyes, Hilyte 405 or Hilyte 647 (AnaSpec Inc) with the proteins for 12 hours at 4°C. Excess dye was removed using PD SpinTrap™ G-25 (Cytiva Life sciences) columns and the labeled and unlabeled protein samples were mixed in 1:99 ratio and used for the experiments. We do not expect the Hilyte dyes to have any discernable effects on our observations as they are comparable to routinely used xanthane dyes. Moreover, to minimize their effects if any, we used only 1% of total protein with labeled ones in our assays. For thioflavin-S (ThS) staining, aliquots of samples from the reactions were centrifuged at 18,000xg for 20 minutes, and pellets were resuspended in 20 mM MES buffer pH 6.0. ThS was added at a final concentration of 10 μM and incubated for 10 minutes prior to imaging.

Dhakal S., Wyant C.E., George H.E., Morgan S.E, & Rangachari V. (2021). Prion-like C-terminal domain of TDP-43 and α-Synuclein interact synergistically to generate neurotoxic hybrid fibrils. Journal of molecular biology, 433(10), 166953.

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Fluorometric Actin Polymerization Assay

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Skeletal rabbit actin was purified and labeled on C374 with N-(1-pyrene)-iodoacetamide. A correction factor was applied for determining pyrene actin concentration, A_corr = A₂₉₀ – (0.127 × A₃₄₄). Actin polymerization was measured in a cuvette-based fluorometer (PTI Quantamaster 400, HORIBA Scientific) with excitation at 365 nm and emission at 407 nm. Gel-filtered G-actin monomers (10% pyrene labeled) were desalted (PD SpinTrap G-25, Cytiva) into a modified G-buffer without ATP [2 mM tris-HCl, 0.1 mM CaCl₂, 1 mM NaN₃, and 1 mM DTT (pH 8.0)], stored on ice, and used within 3 hours. G-actin was converted to the Mg²⁺-bound form by addition of 50 μM MgCl₂ and 0.2 mM EGTA for 2 min at room temperature, before starting the polymerization reaction by mixing G-actin (3× stock) with KMEI buffer (1.5× stock) in a 1:2 ratio, respectively. Wild-type myosin and ADP were included in the 1.5× KMEI buffer, as needed. Final reaction conditions were 2 μM G-actin, 1 μM myosin-15, 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, and 10 mM imidazole (pH 7.0) at 25° ± 0.1°C. Data were corrected for the reaction dead time.

Gong R., Jiang F., Moreland Z.G., Reynolds M.J., de los Reyes S.E., Gurel P., Shams A., Heidings J.B., Bowl M.R., Bird J.E, & Alushin G.M. (2022). Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia. Science Advances, 8(29), eabl4733.

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Fluorescent EGF Receptor Binding Assay

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Fluorescein-labeled EGF (Fluo-EGF) was prepared by mixing 100 μM human recombinant EGF (053-07871; FUJIFILM WAKO Pure Chemical Corporation) and 1 mM fluorescein 5-isothiocyanate (FITC; F0026; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) in 100 mM NaHCO₃ aq for 16 h at 25 °C, followed by the removal of unreacted FITC with PD SpinTrap™ G-25 (28918004; Cytiva, Tokyo, Japan). The proportion of fluorescein-labeled EGF molecules among all molecules was calculated to 0.16.
To immobilize EGFR to the bottom of the microplate, 100 μL of 10 μg/mL human Fc region-fused human recombinant EGFR (10001-H02H; Sino Biological, Inc., Beijing, China) in PBS were added to Pierce™ Protein A Coated Plate (15132; Thermo Fisher Scientific), followed by 1 h of reaction at 25 °C. After three washes with ice-cold PBS, 100 μL of 100 ng/mL Fluo-EGF in PBS were added, followed by reaction for 1 h at 25 °C. After washing with PBS, The fluorescence intensity was measured (Ex 485 nm/Em 526 nm). The signals obtained in the absence and presence of EGFR were defined as 0% and 100% EGFR–EGF binding, respectively.

Sakamoto H., Nishikawa M, & Yamada S. (2024). Development of tight junction-strengthening compounds using a high-throughput screening system to evaluate cell surface-localized claudin-1 in keratinocytes. Scientific Reports, 14, 3312.

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Sequential Protein Extraction from Glue

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Sequential
extraction of proteins was performed: 100 mg of glue or raw material
was mixed with 1000 μL of buffer 1 containing 0.5 M NaCl and
30 mM Tris-HCl and incubated at room temperature for 2 h in the rotator.
Then, samples were centrifuged at 4 °C and 16,000 rpm for 15
min. The supernatant was extracted, desalted with PD-Spintrap G-25
(Cytiva) using 50 mM NH₄HCO₃, and saved for
further proteomics analysis (fraction 1). The pellet was resuspended
in 1000 μL of buffer 2 containing 7 M urea, 2 M thiourea, 4%
3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS),
40 mM Tris, and 75 mM DTT. Extraction 2 was performed at 4 °C
overnight on the shaker. Then, samples were centrifuged at 4 °C
and 16,000 rpm for 15 min, and the supernatant was extracted. CHAPS
was removed from the supernatant using Pierce Detergent Removal Spin
Column, 0.5 mL (Thermo Scientific), following the manufacturer’s
instructions. The supernatant was saved for further proteomics analysis
(fraction 2).

Prisby R., Luchini A., Liotta L.A, & Solazzo C. (2024). Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties. Journal of Proteome Research, 23(5), 1649-1665.

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