Bacteria were resuspended in 150 μL lysis buffer (1% Triton X-100, 0.5% SDS, 1 tablet cOmplete EDTA-free protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) in 10 mL PBS) and lysed by ultrasonication (5 × 20 s, 80%, on ice during breaks). Cell debris was pelletized (5000g, 10 min, 4 °C) and the supernatant was sterile filtered through a 0.2 μm pore size PTFE filter. Protein concentration was determined using a BCA assay (Roti-Quant universal, Carl Roth GmbH + Co. KG), all samples were adjusted to the same volume and concentration (ca. 1 mg mL−1) and transferred to protein low-bind microcentrifuge tubes (Eppendorf, Hamburg, Germany). To precipitate the proteins, 4× sample volume acetone (−80 °C) was added and the samples were stored at −80 °C overnight. The samples were centrifuged at 21 000g at 4 °C for 15 min and the supernatant was discarded. The pellet was resuspended in 500 μL methanol (−80 °C) with ultrasonication (10 s, 10%). After centrifugation at 21 000g and at 4 °C for 15 min, the supernatant was discarded and the pellet was air-dried.
Roti quant universal
Manufactured by Carl Roth
Sourced in Germany
The Roti-Quant universal is a highly versatile and accurate spectrophotometer designed for a wide range of applications in the laboratory. It offers precise measurements of optical density, absorbance, and concentration across various wavelengths, enabling users to analyze a diverse range of samples efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Protein low bind microcentrifuge tubes, by Eppendorf (2 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Myeloperoxidase activity assay kit, by Abcam (1 mentions)
Ab152146, by Abcam (1 mentions)
Rabbit mab igg isotype control, by Cell Signaling Technology (1 mentions)
Protein a g agarose beads, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
7 protocols using roti quant universal
1
Bacterial Protein Extraction and Purification
2
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blotting, cells were washed with PBS and lysed in lysis buffer (100 mM Tris-HCl, pH = 6.8; 20% glycerol; 1% SDS) containing protease and phosphatase inhibitor cocktails. Protein concentration was measured using Roti®-Quant universal (Carl Roth, Karsruhe, Germany) according to the manufacturer’s instructions. Cell lysates were supplemented with bromophenol blue (final concentration 0.01% (w/v)) and β-mercaptoethanol (final concentration 1% (v/v)) prior to being heated for 5 min at 95 °C. Equal amounts of protein (10 μg) were loaded into a 12% polyacrylamide gel, separated by SDS-polyacrylamide gel electrophoresis and subsequently electrotransferred onto nitrocellulose membranes. Reversible Ponceau S. staining was performed to assess equal sample loading. Then, the membranes were blocked with 5% non-fat dry milk in TBST (10 mM Tris-HCl pH = 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20) and appropriate primary and secondary antibodies were used for immunodetection. The proteins were visualized by ECL Plus reagent according to the manufacturer’s instructions. The intensity of bands was semi-quantitatively analysed using the ImageJ software (National Institute of Mental Health, Bethesda, MD, USA).
3
Quantifying Biofilm Biomass on Anodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The dry weight equivalent of the biofilms attached to the anodes was quantified by analysis of the protein content of cell lysate. The individual anodes were placed in 1 mL lysis buffer (LyB, see Supplementary Table 3 ) for at least 24 h. The protein content was then determined with a colorimetric test (Roti-quant universal, Carl Roth, Germany) using a 96-well plate and a plate reader (Tecan Spark, Tecan Austria GmbH, Austria) according to the manufacturers' instructions. The dry weight equivalent was calculated using a MR-1 standard (filter cake from a cell culture with known volume, cell density, and dry weight equivalent). The dry weight equivalent of the planktonic cells m was obtained by {m}mg = 716 · {OD600}.
4
Quantifying Neutrophil Infiltration via MPO
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Myeloperoxidase (MPO) activity was measured as a marker for neutrophil infiltration. The right lungs were homogenized in buffer containing 10 mM NEM (Sigma-Aldrich, Taufkirchen, Germany) and then solubilized in buffer containing 0,5% HTA-Br. MPO activity was determined by the use of Myeloperoxidase activity assay kit (abcam, Cambridge, UK). In addition protein amount was determinated using RotiQuant universal (Roth, Karlsruhe, Germany).
5
Metabolic Profile Analysis of Drosophila
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 10 days of treatment with PBS, CM3, AI, or acarbose, five W1118 flies per sex and treatment were homogenized in 250 μl PBS/Triton X-100 (1%, v/v). Lysates were centrifuged at 12,000 × g and 4°C for 5 min. Supernatants were stored at −80°C until further use. Triglyceride and glucose levels were measured by using commercially available kits (Fluitest TG and GLU; Analyticon Biotechnologies AG, Germany) according to manufacturer's instructions. Samples were diluted 1:2 and 1:5 with PBS/NaCl (0.9%, w/v) for triglyceride and glucose, respectively. Concentrations were calculated via the standard curve and normalized to median fly weights. Measurement of total protein was performed with Roti-Quant Universal (Carl Roth, Germany) according to manufacturer's instructions. The samples were diluted 1:30 with PBS. BSA dissolved in PBS was used as standard. Protein concentrations of the fly samples were calculated via the standard curve and normalized to fly weights.
6
Affinity Purification of c-Myc Interactome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacteria were resuspended in 800 μL co-IP lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5% glycerol, pH 7.4) and 120 μg lysozyme was added. The samples were incubated at 37 °C under shaking at 1400 rpm for 1 h. Afterwards, 8 μL 10% NP-40 solution was added and the bacteria were lysed by ultrasonication (5 × 30 s, 80%, on ice during breaks). The insoluble fraction was pelletized (10 000g, 30 min, 4 °C) and the supernatant was sterile filtered through a 0.2 μm PTFE filter. Protein concentration was determined using a BCA assay (Roti-Quant universal, Carl Roth GmbH + Co. KG). 30 μL Protein A/G agarose beads (Thermo Fisher Scientific) were transferred to protein low-bind microcentrifuge tubes (Eppendorf) and washed with 1 mL co-IP wash buffer (50 mM Tris–HCl, 150 mM NaCl, 5% glycerol, 0.05% NP-40, pH 7.4) and centrifuged for 1 min at 1000g at 4 °C. 500 μg proteome (in 500 μL) and either 1 μL anti-c-Myc antibody (rabbit polyclonal, ab152146, 1 mg mL−1, Abcam) or 0.4 μL rabbit mAb IgG isotype control (2.5 mg mL−1, Cell Signaling Technology, Danvers, United States) were added. The samples were incubated at 4 °C for 3 h under constant rotation. The supernatant was removed after centrifugation (1000g, 1 min, 4 °C), and the beads were washed twice with 1 mL co-IP wash buffer. The detergent was removed by washing the beads twice with co-IP lysis buffer.
7
Determining Encapsulation Efficiency of Coencapsulated Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The encapsulation efficiency (EE, %) of CP was determined using UV/VIS spectrophotometry. To extract free CP from the W 1 /O/W 2 emulsions, the emulsions (6 g) were diluted with demineralized water (6 g) in a 50 mL conical tube and then gently hand-shaken. The diluted emulsions were centrifuged at 7619g and 4 °C for 90 min using a Centrifuge 5430 R (Eppendorf AG, Germany) to separate them into 2 layers: the creamed layer (upper) and the serum layer (bottom). The serum layer from each centrifuged sample was collected using a syringe needle (0.80 × 120 mm) and filtered using a 0.22 μm syringe (33 mm, Rotilabo®-syringe filters, CME, Carl Roth GmbH + Co. KG, Germany) to remove oil droplets. The filtrate was used to detect the free CP. The EE was evaluated by determining the amount of CP in each of the collected filtrates by the BCA protein assay (Roti®-Quant Universal, Carl Roth GmbH + Co. KG, Germany). The EE was indirectly calculated using a calibration curve constructed from a series of CP solutions with standard concentrations. The EE was then obtained as a percentage using eqn (2):
where A 0 is the amount of CP added to the internal aqueous phase, and A is the amount of free CP extracted from the external aqueous phase.
where A 0 is the amount of CP added to the internal aqueous phase, and A is the amount of free CP extracted from the external aqueous phase.
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!