Temed

Manufactured by Thermo Fisher Scientific

Sourced in United States

TEMED is a chemical reagent used in polyacrylamide gel electrophoresis (PAGE) applications. It functions as a catalyst, promoting the polymerization of acrylamide monomers to form the gel matrix.

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34 protocols using temed

Preparation of Gelatine and HEMA Hydrogels

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Gelatine hydrogel (G gel) sheets were prepared in a glass mould. Glutaraldehyde (3% v/w) was added to the solution of gelatine (6% w/w). The solution was placed in the glass mould and frozen in a Julabo cooling chamber at −12 °C for 20 h. It was then defrosted and washed with an excess of water. 2-hydroxyethyl methacrylate gels were prepared by dissolving 2-hydroxyethyl methacrylate (HEMA, Acros Organic, 98%) and poly(ethylene glycol) diacrylate (PEGDA, Aldrich, Mn ~258) in water (6 w/v% solution, HEMA:PEGDA molar ratio 8:1). The reaction mixture was degassed at low pressure for 25 minutes to eliminate dissolved oxygen before gelation. In the conventional method the mixture was cooled to 0 °C for 15 min and then N,N,N′,N′-tetramethyl-ethylenediamine (TEMED, Fisher Scientific, 99%), and ammonium persulfate (APS, 98%) were added and the mixture allowed to freeze completely. In the pre-freezing method the mixture was cooled with constant mixing in an ethanol cooling bath at −18 °C. After ice crystals formed, the mixture was pre-cooled to −2 °C with constant mixing. N,N,N′,N′-tetramethyl-ethylenediamine (TEMED, Fisher Scientific, 99%), and ammonium persulfate (APS, 98%), were added and the mixture allowed to freeze completely. The frozen mixture was kept at −18 °C for 20 hours and then defrosted at room temperature.

Savina I.N., Ingavle G.C., Cundy A.B, & Mikhalovsky S.V. (2016). A simple method for the production of large volume 3D macroporous hydrogels for advanced biotechnological, medical and environmental applications. Scientific Reports, 6, 21154.

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Polyacrylamide Gel Preparation

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Gels are prepared as
described previously.^{16 (link)} A solution of 0.02%
acrylamide in distilled water was degassed until no more bubbling
was visible and the solution was cold to touch. Linear PAA polymerization
is initiated with the addition of 0.024% ammonium per sulfate (APS;
Fisher Scientific) and 0.050% tetramethylethylendiamine (TEMED; Fisher
Scientific). The solution is set at room temperature to polymerize
for 2 h to allow full polymerization of polyacrylamide, yielding a
viscous fluid composed of long linear chains of polyacrylamide.

Swoger M., Gupta S., Charrier E.E., Bates M., Hehnly H, & Patteson A.E. (2022). Vimentin Intermediate Filaments Mediate Cell Morphology on Viscoelastic Substrates. ACS Applied Bio Materials, 5(2), 552-561.

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Fabrication of MA-Alginate Scaffolds

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MA-alginate was synthesized as previously reported with a degree of substitution around 11% [15 ]. MA-alginate scaffolds were prepared by radical polymerization under subzero temperature. For 50 μL of 1 wt% MA-alginate water solution, 0.32 μL of tetramethylethylenediamine (TEMED, Fisher Scientific) and 1.25 μL of 10% ammonium persulfate (APS, Fisher Scientific) water solution were added. The solution was mixed and transferred to a 1-mL syringe and immediately put in a −20°C freezer for at least 12 h to form a disc-shaped scaffold.

Fan Z., Deng J., Li P.Y., Chery D.R., Su Y., Zhu P., Kambayashi T., Blankenhorn E.P., Han L, & Cheng H. (2019). A New Class of Biological Materials: Cell Membrane-Derived Hydrogel Scaffolds. Biomaterials, 197, 244-254.

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Cryogel Alginate Scaffolds for Nanoparticle Loading

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Macroporous MA-alginate scaffolds were prepared via a cryogelation method, in which MA-alginate was chemically crosslinked at −20 °C [39 (link)]. MA-alginate was dissolved in DI water to make a 1% wt solution. For 50 μl of MA-alginate solution, 0.32 μL of tetramethylethylenediamine (TEMED, Fisher Scientific) was added and mixed well with the solution. Then 1.25 μL of 10% ammonium persulfate (APS, Fisher Scientific) water solution was added. After mixing, the solution was transferred to a 1 mL syringe (BD, USA) and immediately placed in a −20 °C freezer for at least 12 h to form a disc-shaped scaffold. Afterwards, the scaffold was thawed and removed from the syringe, washed in 5 mL DI water for 1 h to remove the APS and TEMED used for crosslinking. The scaffold was then lyophilized. In order to load nanoparticles, each scaffold was rehydrated with either 40 μL PLGA NP solution (50 mg/mL in H₂O) or RBCM-PLGA NP solution (50 mg/mL in H₂O) for 30 min. The scaffolds were then lyophilized again and stored for further studies.

Fan Z., Li P.Y., Deng J., Bady S.C, & Cheng H. (2018). Cell membrane coating for reducing nanoparticle-induced inflammatory responses to scaffold constructs. Nano research, 11(10), 5573-5583.

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Oxidative Stress in SH-SY5Y Cells

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Fetal bovine serum (FBS), phosphate buffered saline (PBS), Dulbecco’s minimum essential medium plus F12 (DMEM/F12), penicillin and streptomycin were purchased from Invitrogen (Gaithersburg, MD). Acrylamide/ bis-acrylamide, tris base, glycine, ammonium persulfate, PVDF membrane, TEMED, DTT, SDS, urea, thiourea, glycerol, ammonium bicarbonate, DMSO, ECL reagent, bromoplenol blue were purchased from Fisher Scientific (Pittsburgh, PA). Trypsin and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO).
SH-SY5Y cells were cultured in DMEM/F12 containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂. In order to perform treatments the media were supplemented with 2 mM H₂O₂ (Sigma-Aldrich, MO), 25 μM Edaravone (Abcam, MA) or a combination of H₂O₂/Edaravone for 8 h (Kaste et al., 2013 (link)).

Jami M.S., Salehi-Najafabadi Z., Ahmadinejad F., Hoedt E., Chaleshtori M.H., Neubert T.A., Larsen J.P, & Møller S.G. (2015). Edaravone leads to proteome changes indicative of neuronal cell protection in response to oxidative stress. Neurochemistry international, 90, 134-141.

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Fabrication of Tissue-Mimicking Phantoms

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Stock solutions with a ratio of acrylamide to bis-acrylamide of 37.5:1 (Fisher Scientific, Waltham MA) and acrylamide concentration from 4% to 32% (w/v) in 1× PBS solution were prepared to fabricate tissue mimicking phantoms of different moduli. Titanium dioxide was added as a scattering agent to more clearly define the boundaries between the phantom and samples when performing ultrasound elastography. The polyacrylamide solutions were crosslinked by free radical polymerization using 1.7% and 0.1% (v/v) of tetramethyl ethylenediamine (TEMED, Fisher Scientific, Waltham MA) and ammonium persulfate (APS, Fisher Scientific, Waltham MA), respectively. The phantoms were manufactured in plastic molds with two polyacrylamide gel layers. 100 ml of the above mixture was stirred and poured into the plastic phantom mold to form the first layer. After the first layer gelled, the PDMS samples were placed and lined up on the gelled polyacrylamide. The second 100 ml acrylamide mixture was then poured on top of the first layer, embedding the PDMS samples. The complete PDMS embedded phantom fabrication process can be seen in Figure 1.

Zhou H., Goss M., Hernandez C., Mansour J.M, & Exner A. (2015). Validation of Ultrasound Elastography Imaging for Nondestructive Characterization of Stiffer Biomaterials. Annals of biomedical engineering, 44(5), 1515-1523.

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Oxidative Stress Induction in SH-SY5Y Cells

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Fetal bovine serum (FBS), phosphate buffered saline (PBS), Dulbecco’s minimum essential medium plus F12 (DMEM/F12), penicillin and streptomycin were purchased from Invitrogen (Gaithersburg, MD). Acrylamide/ bis-acrylamide, tris base, glycine, ammonium persulfate, PVDF membrane, TEMED, DTT, SDS, urea, thiourea, glycerol, ammonium bicarbonate, DMSO, ECL reagent, bromoplenol blue were purchased from Fisher Scientific (Pittsburgh, PA). Trypsin and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO).
SH-SY5Y cells were cultured in DMEM/F12 containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂. In order to perform treatments the media were supplemented with 2 mM H₂O₂ (Sigma-Aldrich, MO), 200 μM L-DOPA (Acros Organics, NJ), or a combination of H₂O₂/L-DOPA for eight hours (Table 1).

Jami M.S., Pal R., Hoedt E., Neubert T.A., Larsen J.P, & Møller S.G. (2014). Proteome analysis reveals roles of L-DOPA in response to oxidative stress in neurons. BMC Neuroscience, 15, 93.

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Optimizing Acrylamide Gel Roadblock Fabrication

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A dynamic range of concentrations of acrylamide/bis solution (Bio-Rad, Hercules, CA, USA), TEMED (Fisher Scientific, Waltham, MA, USA), and ammonium persulfate (APS; Thermo Fisher, Waltham, MA, USA) were tested in the device to determine which concentrations leaked and which concentrated the most DNA. A 1x acrylamide/bis and 1x TEMED/APS solution contained 111 μl 30% 29:1 or 19:1 acrylamide/bis solution, 889 μl H₂O, 7.5 μl 10% APS, and 0.8 μl TEMED. This concentration (1×1x) was based on Dimalanta et al. acrylamide gel concentration [9 (link)]. The acrylamide solutions were labeled AxBx-C, where A corresponded to the amount of acrylamide in 1 mL of solution, with A = 1 (111 μl 30% acrylamide), A = 2 (222 μl), etc. B corresponded to the crosslinking agents in the solution, with B = 1 (7.5 μl 10% (w/v) APS and 0.8 μl TEMED) and B = 2 (15 μl 10% APS and 1.6 μl TEMED). C indicated whether the roadblock was made with 19:1 or 29:1 acrylamide-bisacrylamide solution. The Eppendorf tubes were filled with the acrylamide and water and degassed for 15 minutes. Then the APS and TEMED were added and vortexed. Two polydimethylsiloxane (PDMS) dams were positioned in the channel of the 3D printed device to create a region where the acrylamide cured. The acrylamide solution was then loaded into the device and allowed to cure.

Liu K., Schoonmaker M.M., Levine B.L., June C.H., Hodes R.J, & Weng N.P. (1999). Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes. Proceedings of the National Academy of Sciences of the United States of America, 96(9), 5147-5152.

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Immobilization of Bacteriorhodopsin in Polyacrylamide

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Bacteriorhodopsin was immobilized within a 5% polyacrylamide matrix that was buffered with 50 mM TAPS (pH 8.5). The optical density (OD) of each sample was approximately 1 at the absorption maximum of the bR_B state. To prepare the polymer-based cuvettes, the BR solution was first sonicated on ice for 60 seconds using 10-second intervals, to ensure homogeneity, and was then filtered using 5 μm filter paper into a sterile falcon tube. Ammonium persulfate (400 μL) was added, and the solution was degassed for 30 minutes. The protein solution was transferred equally into either 4.0 mL or 1.5 mL methacrylate cuvettes (Plastibrand Cuvettes, Fisher Scientific, Inc.) that were optically transparent on all sides. Next, 0.5% (v v^-1) tetramethylethylenediamine (TEMED; Fisher Bioreagents, electrophoresis grade, assay 97%) was added to each cuvette, mixed well, and allowed to polymerize at ambient temperature. A 15% (w v^-1) polyvinyl alcohol (PVA; Aldrich, 99+% hydrolyzed, avg. MW 89-98,000) solution, which was degassed and buffered with TAPS, was applied to fill the remaining headspace within the cuvette. The cap was sealed using a chemically inert adhesive and was wrapped with Parafilm to prevent condensation within the cuvette.

Ranaghan M.J., Greco J.A., Wagner N.L., Grewal R., Rangarajan R., Koscielecki J.F., Wise K.J, & Birge R.R. (2014). Photochromic Bacteriorhodopsin Mutant with High Holographic Efficiency and Enhanced Stability via a Putative Self-Repair Mechanism. ACS Applied Materials & Interfaces, 6(4), 2799-2808.

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Polyacrylamide Hydrogel Preparation and Compliance Characterization

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Polyacrylamide hydrogels were prepared on glass slides, as detailed previously[17 (link)]. Briefly, premixed acrylamide and bis-acrylamide solution supplemented with ammonium persulfate and tetramethylethylenediamine (TEMED, Invitrogen) was polymerized between aminopropyltrimethoxysilane (APTMS) and glutaraldehydre pretreated glass slide and dichlorodimethylsilane (DCDMS) pre-treated nonreactive glass coverslip. Three different ratios of acrylamide and bis-acrylamide; 300:1, 30:1, and 10:1 were selected to ensure sufficient variation in substrate compliance. Substrate compliance J was defined as the reciprocal of Young’s moduli of >10 separate gels measured by AFM (Veeco). The surface of synthesized hydrogels was coated with UV-activable crosslinker sulfo-SANPAH (Pierce) to bind 0.2 mg/ml type I collagen (BD Biosciences). Unless specified otherwise, all reagents and chemicals were supplied by Sigma Aldrich.

Kim D.H, & Wirtz D. (2015). Cytoskeletal tension induces the polarized architecture of the nucleus. Biomaterials, 48, 161-172.

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