Recombinant human TFAM and AP-DNA were prepared as described previously (23 (link)). Reactions of AP-DNA with TFAM were carried out in the presence of 4 μM AP-DNA, 8 μM TFAM, 20 mM HEPES (pH 7.4), 90 mM NaCl, 20 mM EDTA, without or with 25 mM NaBH3CN. Reaction products were quenched with 0.1 M NaBH4 and digested by trypsin, followed by denaturing PAGE analysis. Proteolytic digestion with trypsin was performed using 60 μg of trypsin (Worthington) per 24 pmol of TFAM at 37°C for 2 h. trypsin digestion was performed with all TFAM-DPC samples to facilitate migration into gels except when assigning different DPC species, whereby trypsin digestion was followed by proteinase K treatment. The digestion with proteinase K was used to convert peptides in peptide-DNA cross-links from trypsin cleavage to smaller peptide fragments. Digestion was performed with 1 unit of proteinase K (NEB, P8107S) per 24 pmol of trypsin-digested TFAM sample for 2 h at 37°C. To examine the thermostability of DPC, products from 1-h and 6-h reactions were heated at 90°C for 10 min before quenching with NaBH4.
Trypsin
Manufactured by Worthington
Sourced in United States, United Kingdom, Germany, Switzerland
Trypsin is a proteolytic enzyme commonly used in cell culture and molecular biology applications. It is responsible for the hydrolysis of peptide bonds, specifically those involving the carboxyl group of lysine or arginine residues. Trypsin is a critical component in the process of cell dissociation, allowing for the detachment of adherent cells from a culture surface.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (26 mentions)
Neurobasal medium, by Thermo Fisher Scientific (14 mentions)
B27 supplement, by Thermo Fisher Scientific (13 mentions)
Glutamax, by Thermo Fisher Scientific (13 mentions)
Collagenase, by Worthington (11 mentions)
Collagenase type 2, by Worthington (10 mentions)
Collagenase type 4, by Worthington (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Dnase, by Merck Group (7 mentions)
Collagenase, by Roche (7 mentions)
Trypsin, by Merck Group (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Tpck treated trypsin, by Worthington (6 mentions)
Dnase 1, by Merck Group (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Trypsin inhibitor, by Merck Group (5 mentions)
Dnase 1, by Worthington (5 mentions)
Poly l lysine (pll), by Merck Group (5 mentions)
Collagenase type d, by Roche (5 mentions)
Dnase, by Worthington (5 mentions)
192 protocols using trypsin
1
TFAM-DNA Cross-Linking and Thermostability
2
Tryptic Digestion and Sample Cleanup
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Urea and TFA eluates were diluted/neutralized with 180 μL 100 mM TEAB. Samples were digested in solution with 1 μg Trypsin (Worthington, LS003740 TPCK-treated Trypsin) over night at 37 C. Digestion was stopped by acidification to final concentration of 1% formic acid.
SDS eluates were prepared following an S-Trap 96-well plate (Protifi LLC, C02-96well) protocol. Eluates were acidified with ~12% phosphoric acid (10:1 v/v) and loaded into the S-trap containing 350 μL 90% MeOH in 100 mM TEAB and spun at 1500x g for 1 min. This step was repeated three times. Then samples were resuspended in 100 μL 100 mM TEAB with 1 μg Trypsin (Worthington) and digested over night at 37 C. Samples were eluted from the S-traps in three consecutive steps, each for 1 min at 1500x g, first with 50 μL 50 mM TEAB, then 50 μL 0.1% TFA and finally 50 μL 50% ACN /0.1% TFA. The combined eluates were dried down in a vacuum centrifuge and resuspended in 2% ACN / 0.1% TFA for LC-MS.
SDS eluates were prepared following an S-Trap 96-well plate (Protifi LLC, C02-96well) protocol. Eluates were acidified with ~12% phosphoric acid (10:1 v/v) and loaded into the S-trap containing 350 μL 90% MeOH in 100 mM TEAB and spun at 1500x g for 1 min. This step was repeated three times. Then samples were resuspended in 100 μL 100 mM TEAB with 1 μg Trypsin (Worthington) and digested over night at 37 C. Samples were eluted from the S-traps in three consecutive steps, each for 1 min at 1500x g, first with 50 μL 50 mM TEAB, then 50 μL 0.1% TFA and finally 50 μL 50% ACN /0.1% TFA. The combined eluates were dried down in a vacuum centrifuge and resuspended in 2% ACN / 0.1% TFA for LC-MS.
3
Protease Accessibility in Sporulating Cells
4
Protease Susceptibility Assay for Forespore Membrane Proteins
5
Protease Susceptibility Assay in Sporulating Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protease susceptibility assays were performed in sporulating cells lacking the SpoIIQ (Q) protein (strain bHC70) to ensure that the membrane proteins present in the inner and outer forespore membranes would not be artificially inaccessible because of protoplast engulfment [65 (link)]. Twenty-five milliliters of sporulating cells (induced by resuspension) were harvested by centrifugation at hour 2.5 after the onset of sporulation, washed, and resuspended in 2 mL 1X SMM buffer (0.5 M sucrose, 20 mM MgCl2, 20 mM maleic acid, pH 6.5). The cells were protoplasted by lysozyme (5 mg/mL final concentration) for 10 min with slow agitation. The protoplasts were harvested by centrifugation and resuspended in 1 mL of 1X SMM. Protoplasts (100 μL) were incubated with trypsin (30 μg/mL final concentration) (Worthington), trypsin and Triton X-100 (2% final concentration), or 1X SMM for 2 h at 30°C. Reactions were terminated by the addition of 100 μL of 2X SDS-sample buffer and incubation for 5 min at 95°C. Five microliters from each reaction were analyzed by immunoblot.
6
Trypsin Digestion of MutLγ Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trypsin digestions were performed in 30 μl reactions with 2 μM MutLγ in 25 mM HEPES-NaOH pH 7.5, 15 mM CaCl2, 100 mM NaCl, 3% glycerol, with or without 5 mM ATP (sodium salt, pH 7) with 25 ng Trypsin (Worthington). After 5 minutes digestion at room temperature, reactions were stopped in Laemmli sample buffer. Proteins were separated on 10% NuPAGE Bis:Tris gels in MOPS SDS running buffer and visualized by Coomassie staining.
7
Protein Digestion and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Urea and TFA eluates were diluted/neutralized with 180 μL 100 mM TEAB. Samples were digested in solution with 1 μg Trypsin (Worthington, LS003740 TPCK-treated Trypsin) over night at 37 C. Digestion was stopped by acidification to final concentration of 1% formic acid.
SDS eluates were prepared following an S-Trap 96-well plate (Protifi LLC, C02-96well) protocol. Eluates were acidified with ∼12% phosphoric acid (10:1 v/v) and loaded into the S-trap containing 350 μL 90% MeOH in 100 mM TEAB and spun at 1,500x g for 1 min. This step was repeated three times. Then samples were resuspended in 100 μL 100 mM TEAB with 1 μg Trypsin (Worthington) and digested over night at 37 C. Samples were eluted from the S-traps in three consecutive steps, each for 1 min at 1,500 × g, first with 50 μL 50 mM TEAB, then 50 μL 0.1% TFA and finally 50 μL 50% ACN/0.1% TFA. The combined eluates were dried down in a vacuum centrifuge and resuspended in 2% ACN/0.1% TFA for LC-MS.
SDS eluates were prepared following an S-Trap 96-well plate (Protifi LLC, C02-96well) protocol. Eluates were acidified with ∼12% phosphoric acid (10:1 v/v) and loaded into the S-trap containing 350 μL 90% MeOH in 100 mM TEAB and spun at 1,500x g for 1 min. This step was repeated three times. Then samples were resuspended in 100 μL 100 mM TEAB with 1 μg Trypsin (Worthington) and digested over night at 37 C. Samples were eluted from the S-traps in three consecutive steps, each for 1 min at 1,500 × g, first with 50 μL 50 mM TEAB, then 50 μL 0.1% TFA and finally 50 μL 50% ACN/0.1% TFA. The combined eluates were dried down in a vacuum centrifuge and resuspended in 2% ACN/0.1% TFA for LC-MS.
8
Isolation and Culture of Hippocampal Neurons from CD1 Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The animal welfare committee of the University of Würzburg, in accordance with European Union guidelines, approved all experimental procedures. Hippocampal neurons were prepared from CD1 mice of either sex, as described earlier [39 (link)]. Hippocampi of newborn mice were collected in Hank’s buffered saline solution (HBSS). Trypsin (Worthington) was added to a final concentration of 0.1% and the tissue incubated for 15 min at 37°C. The protease digestion was stopped with 0.1% Trypsin inhibitor (Sigma). After four steps of trituration in Neurobasal/B27 medium (Life Technologies), cells were plated on poly-L-lysine-coated glass coverslips in Neurobasal, 1× B27, 0.5% penicillin/streptomycin, 1% Glutamax, and 1× N2 supplement (all Life Technologies) and cultured at 37°C under an atmosphere of 5% CO2. Calcium imaging experiments were performed after indicated days in vitro (DIV).
9
Isolation and Culture of CSMG Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CSMG neurons were isolated 48 hr post-sepsis induction. The rats were first anesthetized with CO2 and then quickly decapitated with a laboratory guillotine. The CSMG tissue was removed and cleared of connective tissue in ice-cold Hank’s balanced salt solution (Sigma-Aldrich Chemical, St. Louis, MO). The ganglia were enzymatically dissociated in a modified Earle’s balanced salt solution containing 0.6 mg/ml collagenase (Roche Pharmaceuticals, Switzerland), 0.4 mg/ml trypsin (Worthington Biochemical, Lakewood, NJ), and 0.1 mg/ml DNase (Sigma Chemical) in a shaking water bath at 35°C for 60 min. The neurons were dispersed by shaking, centrifuged twice for 6 min at 63 X g, and resuspended in minimal essential medium (MEM), supplemented with 10% fetal calf serum (MidSci, St. Louis, MO), 1% glutamine and 1% penicillin-streptomycin (both from Life Technologies, Carlsbad CA). Finally, the dissociated CSMG neurons were plated onto 35 mm poly-L-lysine coated dishes and stored overnight in a humidified incubator (5% CO2/95% air) at 37°C.
10
Primary Rat Hippocampal Neuron Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hippocampi were dissected from E18.5 Wistar rat embryos (Charles River Laboratories, United Kingdom), treated with trypsin (Worthington, United Kingdom) at 0.5 mg/ml and triturated with fire-polished Pasteur pipettes. Dissociated cells (80–100k) were plated onto 35 mm low profile Grid500 μ-dishes (Ibidi, Germany) pre-treated with 100 μg/mL poly-D-lysine (Sigma, United Kingdom) and 10 μg/mL laminin (Thermo Fisher Scientific, United Kingdom). Neurons were maintained in Neurobasal media supplemented with 2% B27, 2% foetal bovine serum, 1% glutamax, and 1% penicillin/streptomycin (all Thermo Fisher Scientific, United Kingdom) in a 37°C humidified incubator with 5% CO2. After 3 days in vitro (DIV) the media was exchanged for serum and antibiotic-free media.
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!