For each biological condition, two 6-well plates of HeLa CCL-2 cells were grown to confluency. Per condition, three independent biological replicates were performed. Cells were infected for 30 or 45 min as described above. The plates were put on ice and washed twice with ice-cold PBS. Samples were then collected in 8 M urea (AppliChem), 0.1 M ammoniumbicarbonate (Sigma-Aldrich), 0.1% RapiGest (Waters), and PhosSTOP (Roche). The samples were briefly vortexed, sonicated at 4°C (Hielscher), shaken for 5 min on a thermomixer, and centrifuged for 20 min at 4°C and 169,000 g. Supernatants were collected and stored at −80°C for further processing. BCA protein assay (Pierce) was used to measure protein concentration.
Urea
Manufactured by AppliChem
Sourced in Germany
Urea is a chemical compound with the formula CO(NH2)2. It is a colorless, odorless, crystalline solid that is highly soluble in water. Urea is a primary component of urine in mammals and is used in a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Chaps, by AppliChem (2 mentions)
Rapigest, by Waters Corporation (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Pbs tween 20, by Merck Group (1 mentions)
Pbs t, by Merck Group (1 mentions)
Silica beads, by Carl Roth (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Chaps, by Roche (1 mentions)
Thiourea, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Tween 20, by Carl Roth (1 mentions)
Hepes, by AppliChem (1 mentions)
Cacl2, by AppliChem (1 mentions)
L arginine, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
7 protocols using urea
1
HeLa Cell Infection Time-course Protocol
2
Phage Display Screening of Nanobodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Receptor binding pockets of rec-NadA–NadA-gdA33−K69 and NadA-ccL121−K158 was recently identified in our previous publication (Mertinková et al., 2020 (link)). The synthetic analogs of peptides–NadA-gdA33−K69 and NadA-ccL121−K158 containing biotin tag at C-terminus (Supplementary Table 1 ) were dissolved in 5 m Urea (AppliChem, Germany), captured separately on neutravidin coated magnetic beads and incubated with VHH-phage library (1 × 1014 Phages) diluted in 1X PBS (1.5 mL) for 1 h at 4°C with constant shaking. Next, the tubes were placed on the magnetic separator, flow-through was discarded and beads were washed with PBS-Tween 20 (0.1%, Sigma Aldrich) (PBS-T). The second washing was performed at 4°C for 16 h. Nine more washings were performed with PBS-T for 5 min and with a change of tubes at every wash. Finally, phages were eluted in 100 mM glycine–HCl (pH 2.7, MikroChem spol. SRO, Bratislava), and pH was raised to 7.5 immediately with Tris (5M, AppliChem). The single round of biopanning was deliberately performed to preserve the diversity of phages and increase the chances of retaining blocking nanobodies.
3
Protein Extraction and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, 1.2 mL of sample was added to a 2-mL reaction tube containing 1 g of silica beads (0.5 mm, Carl Roth, N030.1) and lysed using a bead mill (30 min, 30 Hz) with a subsequent centrifugation step (8000×g, 10 min, room temperature). To a new reaction tube, 1 mL of supernatant was transferred, and 1 mL 20% trichloroacetic acid (w/v, Sigma-Aldrich, 99%) was added before the tube was stored for 1 h at 4 °C. Samples were centrifuged (16,400×g, 10 min, 4 °C, same for following steps) and precipitated twice (alternating) with ice-cold acetone (80% v/v, VWR, 99.8%) and ethanol (70% v/v, VWR, 99.8%) for 20 min with subsequent centrifugation after each step. The dried samples were resuspended afterward in 200 μL urea buffer (8 M urea (Applichem) in 0.1 M Tris–HCl, pH 8.5).
4
Immobilized pH Gradient Proteomics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immobilised pH gradient (IPG) strips and IPG buffers were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Acrylamide/piperazine-di-acrylamide (PDA) solution (37.5:1, w/v) was purchased from Biosolve Ltd. (Valkenswaard, The Netherlands), and the other reagents for the polyacrylamide gel preparation were acquired from Bio-Rad Laboratories. CHAPS was obtained from Roche Diagnostics (Mannheim, Germany), urea from AppliChem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland), 1,4-dithioerythritol (DTE) and EDTA from Merck (Darmstadt, Germany) and tributylphosphine (TBP) from Pierce Biotechnology (Rockford, IL, USA). All reagents were kept at 4°C.
5
Protein Solubilization and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The precipitates of 1 I cell culture supernatant/conditioned media were incubated in 200 μl DIGE lysis buffer (30 mM Tris–HCl buffer, pH 8.5, containing 7 M urea (Applichem, Darmstadt, Germany), 2 M thiourea (Sigma-Aldrich) and 4% (w/v) 3 [(3 cholamidopropyl)-dimethylamino] 1 propane sulfonate (CHAPS; Applichem)) at 25°C for 30 min thereby vortexing thoroughly. Solubilized proteins were then sonicated using two cycles of five impulses (0.5 s/impulse) at 100% power (Bandelin UW 2070 sonicator, MS 73 needle; Bandelin, Berlin, Germany) and total protein concentration of samples was determined as previously described [52 (link)].
6
Recombinant Protein Expression Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following chemicals were bought from Merck Millipore (Darmstadt, Germany): MgSO4, ammonium-acetate, citric acid, KCl, Na2HPO4, KH2PO4, HCl, and EDTA. The following chemicals were bought from Applichem (Darmstadt, Germany): Triton X100, Guanidine, Glutathione, HEPES, CaCl2, Urea, and NaCl. The following chemicals were purchased from Sigma-Aldrich (Vienna, Austria): Tris-Base, β-mercaptoethanol, L-Arginine, and formic acid. Tween-20 was obtained from Roth (Karlsruhe, Germany), acetonitrile from VWR Chemicals (Radnor, PA, USA) and imidazole from NeoLab Migge (Heidelberg, Germany). The pHLsec plasmids were kindly provided by A. R. Aricescu (Cambridge, UK).
7
Extracellular Vesicle Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One and a half million AF-MSCs and HPL cells were cultured in their specific media, as it is described in the previous section. At 80% confluency the media were removed and the cell layers were washed 3 times with 1xPBS (Gibco BRL, Grand Island, NY) and 1 time with serum and phenol red free medium (SFM) (Gibco-Invitrogen, Grand Island, New York). Cells were then incubated with SFM for 24 h and then conditioned media (CM) were collected as previously described [28 (link)]. The latter was centrifuged at 1000 ×g for 10 min at 4 °C to remove dead cells and large debris and incubated with 7.5% Trichloro Acetic Acid (TCA) (Fluka, Buchs Switzerland), 0.1% NLauroyl Sarcosine (NLS) (Fluka) at −20 °C overnight. Centrifugation then followed at 10,000 ×g for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed in ice cold Tetra Hydro Furan (THF) (Sigma-Aldrich Ltd.) and centrifuged again as previously. The final pellet was air dried and resuspended in sample buffer [7 M urea (Applichem, Darmstadt, Germany), 2 M thiourea (Fluka), 4% CHAPS (Applichem), 1% DTE(Sigma), 2% IPG buffer (BioRad) and 3.6% Protease inhibitors (BioRad)] followed by 30 min bath sonication. Samples were stored at −80 °C until use.
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!