Each sample was collected in triplicate and the collected samples were freeze dried (Alpha 2–4-LSC, Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Five milligrams (5 mg) of each of the lyophilized samples was weighed using dual range analytical balance (Model XA105, Mettler Toledo, USA) and transferred to a 2 mL Eppendorf tube. A steal bead of 5 mm in diameter was inserted to each Eppendorf tube and the samples were pulverized in a mixer mill (Model MM 301, Retsch GmbH, Germany) at 25 s−1 for 50 s. Two hundred microliters (200 µL) of extraction solvent [a mixture of water and 10 mM norvaline (1 mL: 5 µL)] was added to each sample and the samples were mixed thoroughly for 20 min on a vortex (JK Janke and Kunkel IKA, model IKA VIBRAX-VXR). The samples were then centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 5 min and the supernatant was transferred to a 1.5 mL Eppendorf tube. This solution was again centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 5 min and the supernatant was transferred to a new 1.5 mL Eppendorf tube. This solution was stored in deep freezer at − 80 °C until the next process.
Model 5415c
Manufactured by Eppendorf
Sourced in Germany
The Eppendorf Model 5415C is a centrifuge that provides reliable and consistent performance for a variety of laboratory applications. It features a compact design, a robust and durable construction, and is capable of reaching a maximum speed of 14,000 rpm.
Automatically generated - may contain errors
6 protocols using model 5415c
1
Metabolite Extraction and Sample Preparation
2
Fmoc Derivatization for Amino Acid Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After thawing at room temperature, 25 µL of the standard and sample solutions were transferred to 1.5 mL Eppendorf tubes. Fifty microliters (50 μL) of 0.5 M sodium borate buffer pH 7.9 and 100 μL 6 mM Fmoc-Cl solution (in acetone) were added to each solution and the resulting mixture was incubated for at least 5 min after mixing. Five hundred microliters (500 μL) of n-pentane was added to each solution, mixed thoroughly, centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 1 min and the upper (organic phase) was discarded. This step was repeated two more times. After the last extraction step and removal of the organic phase, the tubes were opened and allowed to stand in a fume hood for evaporation of any residual organic solvent.
3
Solubilizing Inclusion Body Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 6
Solubilization of Inclusion Bodies. Six mL of inclusion body suspension (containing 32 mg/mL of DIG-657 protein) were centrifuged on the highest setting of an Eppendorf model 5415C microfuge (approximately 14,000×g) to pellet the inclusions. The storage buffer supernatant was removed and replaced with 25 mL of 100 mM sodium carbonate buffer, pH11, in a 50 mL conical tube. Inclusions were resuspended using a pipette and vortexed to mix thoroughly. The tube was placed on a gently rocking platform at 4° C. overnight to extract the target protein. The extract was centrifuged at 30,000×g for 30 min at 4° C., and the resulting supernatant was concentrated 5-fold using an Amicon Ultra-15 regenerated cellulose centrifugal filter device (30,000 Molecular Weight Cutoff; Millipore, Billerica, Mass.). The sample buffer was then changed to 10 mM CAPS [3-(cyclohexamino)1-propanesulfonic acid] pH 10, using disposable PD-10 columns (GE Healthcare, Piscataway, N.J.).
4
Solid-phase Extraction and Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Solid-phase extraction (SPE) was conducted using Chromabond Multi 96-well plate (Macherey–Nagel, Düren, Germany) containing 50 mg/well HR-X-resin (Macherey–Nagel, Düren, Germany). First, the SPE plate was conditioned by 1 mL of methanol followed by 1 mL of water. In this and all subsequent steps, the liquid was passed through the resin by centrifugation at 500×g for 5 min using JS5.3 swingout rotor in an Avanti J-26XP centrifuge (Beckman Coulter, Fullerton, CA, USA). 500 µL of 5% (v/v) acetonitrile was added to the sample and standard solutions mentioned in “Sample derivatization and processing ”. Then, the resulting solutions were quantitatively loaded onto the SPE plate, washed with 1 mL of water and the flow through was discarded after centrifugation. In the next step, 1 mL of methanol was added into the 96-deep well plate and eluted to a new block by centrifugation. Finally, the eluates were transferred from the 96-deep well block to 2 mL Eppendorf tubes, and allowed to evaporate under vacuum in an Eppendorf Concentrator (Model 5301, Eppendorf, Hamburg, Germany) at 45 °C for 45 min. Finally, the samples were centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 10 min and the supernatant was transferred to the 96-well plate and the plate was placed in LC–MS/MS auto-sampler.
5
Vibrio diazotrophicus Challenge in Sea Urchins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sea urchins were challenged with an initial injection of 1 X 104 heat-killed Vibrio diazotrophicus per ml of coelomic fluid (CF, volume per animal was estimated according to [40 (link)] and injected a second or a third time at 24 and 48 hr with 1 X 106 heat-killed V. diazotrophicus per ml of CF as described [53 (link)]. Animals were sacrificed 24 hr after the last injection by removing Aristotle’s Lantern and collecting the wCF in 50 ml tubes on ice, which were gently inverted several times to allow the wCF to clot. The wCF was centrifuged (Eppendorf, model 5415C) at 10,000 x g at 4°C for 5 min and the supernatant, or cell free CF (cfCF), was stored at -70°C in 1 ml aliquots for use in opsonization assays (see below). The pellet was either used to isolate SpTrf proteins (see below) or was discarded.
6
Starch Analysis via Amiloglucosidase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For starch analysis, 50 μg of each sample were ground to a fine powder and suspended in 500 μL of acetate buffer. Samples were carefully mixed and centrifuged at 15000 rpm (Model 5415 C, Eppendorf, Milan, Italy) and the resulting supernatant was used to establish the basal concentration of glucose (the enzymatic method described above). Samples were then incubated at 55°C and 1 mM maltose was added to each. The glucose released by the hydrolysis of maltose due to native wood amiloglucosidases was measured after 5, 10, 30, 60 and 90 minutes, in order to define the linearity (inflexion point approx. at 60 minutes). In vitro amiloglucosidase activity was evaluated by subtracting the basal concentration of glucose from the glucose value measured after 30 minutes. Amiloglucosidase activity was expressed as hydrolyzed maltose (µmol min -1 g - 1 FW).
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!