We obtained the materials from Sigma-Aldrich (Saint Louis, MO, USA): bovine BC, a-LA, b-LG (purity ≥ 98%) and IgG (≥95%) protein standards, human milk LF protein standard (>95%), 1,4-dithiothreitol (DTT), iodoacetamide, acetonitrile, formic acid, and thermolysin (Type X, E.C. No. 3.4.24.27). Tris base, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), ammonium persulfate, N, N, N, N’-tetramethylethylenediamine (TEMED), Precision Plus ProteinTM standards, and colloidal Coomassie G-250 stain, all molecular biology grade, were acquired from Bio-Rad (Hercules, CA, USA). Acrylamide, N, N’-methylenebisAcrylamide, urea, 2-mercaptoethanol, glycine and bromophenol blue, and ultrapure Bioreagent grade, as well as isobutyl alcohol, glacial acetic acid, and sodium hydroxide, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Thermo Fisher Scientific provided Invitrogen’s Qubit® Protein Assay kit. Zip Tips C18 were obtained from Millipore Sigma (Saint Louis, MO, USA). Deionized water, used in all experiments, was purified using a Milli-Q system from Millipore Merck (Darmstadt, Germany).
N n n n tetramethylethylenediamine
Manufactured by Bio-Rad
Sourced in United States
N,N,N′,N′-tetramethylethylenediamine is a chemical compound used as a laboratory reagent. It functions as a buffer and catalyst in various biological and chemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium persulfate, by Bio-Rad (6 mentions)
Bis acrylamide, by Bio-Rad (5 mentions)
Acrylamide, by Bio-Rad (5 mentions)
Z vad fmk, by Alexis Biochemicals (2 mentions)
Glacial acetic acid, by Thermo Fisher Scientific (1 mentions)
Acrylamide, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (1 mentions)
Tris base, by Bio-Rad (1 mentions)
Ethylenediaminetetraacetic acid, by Bio-Rad (1 mentions)
Precision plus proteintm standard, by Bio-Rad (1 mentions)
Urea, by Thermo Fisher Scientific (1 mentions)
Thermolysin, by Merck Group (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
1 4 dithiothreitol, by Merck Group (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
7 protocols using n n n n tetramethylethylenediamine
1
Quantitative Milk Protein Analysis
2
Fabrication of FN-PA Hydrogels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FN-PA hydrogels with varying bisacrylamide concentrations were fabricated on amino-silanized coverslips, as previously described (Brown et al., 2013 (link)). In brief, PA gel solutions were produced by combining acrylamide and bisacrylamide (Bio-Rad) to final concentrations of 8% acrylamide and 0.045%, 0.102%, 0.146%, or 0.239% bisacrylamide to obtain gels with final elastic moduli of 1.8 kPa, 6.7 kPa, 10.6 kPa, or 18.7 kPa, respectively. 35 µl of each solution was polymerized by the addition of 1% (vol/vol) ammonium persulfate (VWR) and 0.1% (vol/vol) N,N,N′,N′-tetramethylethylenediamine (Bio-Rad). Human plasma FN was purified from blood plasma and covalently attached to the surface using 0.2 mg/ml UV-activated heterobifunctional cross-linker sulfosuccinimidyl-6-(4′-azido-2′nitrophenyl-amino)hexanoate (Pierce Chemical). After overnight incubation with 20 µg/ml FN, gels were then washed and stored in PBS.
3
Preparation of Alkylphospholipid Analogs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Edelfosine was obtained from R. Berchtold (Biochemisches Labor, Bern, Switzerland). A stock solution was prepared at 2 mM in RPMI-1640 culture medium, containing 10% (v/v) fetal bovine serum (FBS), by heating at 50°C for 30 min, as previously described [25 (link)]. Perifosine (octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate) and erucylphosphocholine ((13Z)-docos-13-en-1-yl 2-(trimethylammonio)ethyl phosphate) were from Zentaris. Miltefosine (hexadecylphosphocholine) was from Calbiochem. Stock sterile solutions of the distinct APL analogs (2 mM) were prepared in RPMI-1640 culture medium (Invitrogen), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO-BRL), as above. Caspase-4 inhibitor z-LEVD-fmk and the pan-caspase inhibitor z-VAD-fmk were from Alexis Biochemicals. Caspase-3 inhibitor Ac-DEVD-CHO, caspase-9 inhibitor z-LEHD-fmk, and the JNK inhibitor SP600125 were from Calbiochem. Acrylamide, bisAcrylamide, ammonium persulfate, and N,N,N'N'-tetramethylethylenediamine were from Bio-Rad. All other chemicals were from Merck or Sigma.
4
Preparation of Stock Solutions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stock solutions of 100 mM of compounds were made in dimethyl sulfoxide (DMSO) and aliquots were frozen at −20 °C. Poly(vinylidene difluoride) (PVDF) membranes were purchased from Millipore (Billerica, MA). Acrylamide, bisAcrylamide, ammonium persulfate and N, N, N′, N′-tetramethylethylenediamine were from Bio-Rad (Hercules, CA). All other chemicals were obtained from Sigma (Saint Louis, MO).
5
Arginase-1 Inhibition and Caspase Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
If not otherwise stated, chemicals were purchased from Sigma-Aldrich (St Louis, MO). Nor-NOHA was from Cayman Chemical (Ann Arbor, MI). Polyclonal rabbit anti-rat arginase-1 antiserum8 (link), which is cross-reactive to mouse and human arginase-1, was kindly provided by Dr. M. Modolell (Max-Planck Institut for Immunobiology, Freiburg, Germany). Edelfosine was obtained from R. Berchtold (Biochemisches Labor, Bern, Switzerland) and stock solutions were prepared as previously described62 (link). Caspase-4 inhibitor z-LEVD-fmk, and the broad pan-caspase inhibitor z-VAD-fmk were from Alexis Biochemicals (San Diego, CA). Caspase-8 inhibitor z-IETD-fmk was from Calbiochem (San Diego, CA). Acrylamide, bisAcrylamide, ammonium persulfate, and N,N,N′N′-tetramethylethylenediamine were from Bio-Rad (Hercules, CA). RPMI 1640 culture medium without arginine was purchased from GIBCO-BRL (Gaithersburg, MD), and supplemented with MnCl2 to a physiological concentration (4 µM).
6
Modulation of Extracellular Matrix Stiffness
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Poly-acrylamide (PA) gels of varying bisacrylamide concentrations were created on amino-silanated coverslips as previously described [68] . PA gel solutions were produced by combining acrylamide and bisacrylamide to final concentrations of 8% acrylamide (Biorad, Hercules, CA, USA) and 0.048%, 0.117%, 0.208%, or 0.260% bisacrylamide (Biorad) to obtain gels with final elastic moduli of 2 kPa, 8 kPa, 16 kPa, or 24 kPa, respectively. Fifty (50) µl of each solution was polymerized by the addition of ammonium persulfate (VWR, West Chester, PA, USA) and N,N,N′,N′-tetramethylethylenediamine (Biorad, Hercules, CA, USA) (1% and 0.1% final concentration respectively). The gels were allowed to polymerize for approximately 30 minutes, then washed three times with PBS. Fn was covalently attached to the surface using the heterobifunctional crosslinker sulfosuccinimidyl-6- (4′-azido-2′ nitrophenyl-amino) hexanoate (sulfo-SANPAH; Pierce Chemical Co., Rockford, IL., USA). Following an overnight incubation with the Fn, gels were washed three times with PBS.
7
Fabrication of Tunable Polyacrylamide Hydrogels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coverslips were exposed to heat (1080 °F), treated with NaOH (0.1 n ), and activated with glutaraldehyde (0.5% v/v). Next, a prepolymer solution was prepared to contain acrylamide (A) (40%; Bio‐Rad Laboratories), N,N′‐methylene‐bis‐acrylamide (B) (2%; Bio‐Rad Laboratories), 10% ammonium persulfate (Bio‐Rad Laboratories), N,N,N′,N′‐tetramethylethylenediamine (Bio‐Rad Laboratories), and HEPES (VWR). For 0.2 kPa gels, 3% A/0.2% B was used while 8% A/0.6% B was used for 12 kPa PAM gel. The prepolymer solution was deposited on the activated coverslips and allowed to polymerize for 30 min. To coat the polyacrylamide surface with ECM molecules, hydrogels were treated with sulfosuccinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate (sulfo‐SANPAH) (1 mg mL−1), exposed to UV light (wavelength 254 nm) for 15 min, and incubated with rat tail Collagen I or Collagen I‐FITC (20 µg mL−1) at 37 °C for 1 h. The fluorescence intensity of deposited Collagen I‐FITC was measured using ImageJ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!