Cells were inoculated into 10 mL LB containing antibiotics and 0.5 mM IPTG by diluting 1000-fold from the OD600 = 1 suspensions. Cells were grown at 37°C to OD600 ~1, washed twice with 10 mL LB, and then pelleted in 1.5 mL eppendorf tubes at amounts equivalent to 1 mL X 3 OD600. The pellets were re-suspended in 900 μL lysis buffer (50 mM HEPES-KOH, pH 8, 100 mM KOAc, 10% glycerol, 1 mM DTT, 1 mM Phenylmethylsulfonyl fluoride, and protease inhibitor cocktail), incubated with 1 mg/mL lysozyme at room temperature for 30 min, and digested with DNAaseI (50 μg/mL in 16 mM MgCl2) on ice for 10 min. Lysed cells were sonicated in a room temperature bath sonicator. Cell debris and unbroken cells were removed by centrifugation at 2 k rpm, 5 min in Microfuge 18 (Beckman Coulter). The total lysate sample (T) was taken from the supernatant. The inclusion body (I) was isolated by additional centrifugation at 4 k rpm for 5 min. The soluble (S) and membrane (M) fractions were further separated by ultracentrifugation at 48 k rpm for 1 hr in a TLA120.2 rotor (Optima TLX, Beckman Coulter). The inclusion body and membrane samples were dissolved in 5% SDS buffer. All fractions were analyzed using SDS-PAGE and western blotting against the His6-tag.
Microfuge 18
Manufactured by Beckman Coulter
Sourced in United States
The Microfuge 18 is a compact, high-performance benchtop centrifuge designed for versatile laboratory use. It features a maximum speed of 18,000 rpm and a maximum relative centrifugal force of 30,130 xg, enabling efficient sample preparation and separation. The Microfuge 18 accommodates a variety of rotor configurations to handle different tube sizes and volumes.
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Lab products found in correlation
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Optima tlx, by Beckman Coulter (1 mentions)
Thermocycler, by Analytik Jena (1 mentions)
Cm003, by Thermo Fisher Scientific (1 mentions)
Megafuge 8r, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Mouse anti camkiiβ, by LifeSpan BioSciences (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Novex tbe urea gel, by Thermo Fisher Scientific (1 mentions)
E gel ex, by Thermo Fisher Scientific (1 mentions)
Novex tbe urea sample buffer, by Thermo Fisher Scientific (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Allegra x 15r, by Beckman Coulter (1 mentions)
7 protocols using microfuge 18
1
Protein Fractionation and Analysis
2
Bacterial Cell Lysis and PCR Amplification
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Biomass from purified bacteria cultures on nutritive agar (Oxoid CM003) was transferred to tubes containing 10 mL of 0.3% saline sterile solution to achieve a 5 McFarland Standard cell suspension; then, 1 mL was transferred to microcentrifuge tubes (Microfuge 18 Beckman) and centrifuged for 5 min at 10,000 rpm.
The recovered cells were washed with the saline solution. For cell lyses, the suspensions were frozen at -20°C and then heated at 100°C for 10 min. The suspension obtained was immediately centrifuged. After 5 min at 10,000 rpm, 1 μL of lysed cell preparation was used in a PCR reaction with the Primer CsM13 [25 (link)]. The amplification was performed in a thermocycler (Biometra), according to Chambel et al. [26 (link)].
Visualization of PCR products was performed in a horizontal electrophoresis chamber (Pharmacia) with an Electrophoresis Power Supply EPS 600. DNA fragments were stained with 0.2 mg/mL of ethidium bromide and visualized with an ultraviolet transilluminator and Image system Alliance 4.7 Uvitec Cambridge. Data images were analyzed with the BioNumerics 6.6 software.
The recovered cells were washed with the saline solution. For cell lyses, the suspensions were frozen at -20°C and then heated at 100°C for 10 min. The suspension obtained was immediately centrifuged. After 5 min at 10,000 rpm, 1 μL of lysed cell preparation was used in a PCR reaction with the Primer CsM13 [25 (link)]. The amplification was performed in a thermocycler (Biometra), according to Chambel et al. [26 (link)].
Visualization of PCR products was performed in a horizontal electrophoresis chamber (Pharmacia) with an Electrophoresis Power Supply EPS 600. DNA fragments were stained with 0.2 mg/mL of ethidium bromide and visualized with an ultraviolet transilluminator and Image system Alliance 4.7 Uvitec Cambridge. Data images were analyzed with the BioNumerics 6.6 software.
3
Lipid Extraction and Fractionation Protocol
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Capillary
electrophoresis fractions were centrifuged at 18 000g at 4 °C for 1 h (Beckman Microfuge 18). The supernatant
was discarded, and the pellet (very small) was resuspended in methanol
with 0.516 mg/mL butylated hydroxytoluene (BHT). The resuspended pellet
was transferred to a glass tube to minimize contact with plastic,
and methyl tert-butyl ether (MTBE) was added, maintaining
a 10:3 ratio per volume of MTBE/methanol. Extraction was left shaking
overnight (∼16 h) at 4 °C. Extractions were treated with
0.15 M ammonium acetate for phase separation while maintaining a 20:6:5
ratio per volume of MTBE/methanol/ammonium acetate. Extractions were
centrifuged at 2000g at 4 °C for 10 min (Thermo
Fisher Megafuge 8R), and the upper organic phase was collected in
a separate glass tube. Extraction containers were washed with a 20:6:5
ratio per volume of MTBE/methanol/ammonium acetate, centrifuged, and
the organic phase was added to the first collection. Organic phase
samples were completely dried, utilizing speed vacuum at a temperature
of 37 °C; the dried samples were stored at −80 °C
and resuspended in 1:1 chloroform/methanol for mass spectrometry analysis.
The aqueous phase was used for liposome flotation assay and protein–lipid
overlay assay.
electrophoresis fractions were centrifuged at 18 000g at 4 °C for 1 h (Beckman Microfuge 18). The supernatant
was discarded, and the pellet (very small) was resuspended in methanol
with 0.516 mg/mL butylated hydroxytoluene (BHT). The resuspended pellet
was transferred to a glass tube to minimize contact with plastic,
and methyl tert-butyl ether (MTBE) was added, maintaining
a 10:3 ratio per volume of MTBE/methanol. Extraction was left shaking
overnight (∼16 h) at 4 °C. Extractions were treated with
0.15 M ammonium acetate for phase separation while maintaining a 20:6:5
ratio per volume of MTBE/methanol/ammonium acetate. Extractions were
centrifuged at 2000g at 4 °C for 10 min (Thermo
Fisher Megafuge 8R), and the upper organic phase was collected in
a separate glass tube. Extraction containers were washed with a 20:6:5
ratio per volume of MTBE/methanol/ammonium acetate, centrifuged, and
the organic phase was added to the first collection. Organic phase
samples were completely dried, utilizing speed vacuum at a temperature
of 37 °C; the dried samples were stored at −80 °C
and resuspended in 1:1 chloroform/methanol for mass spectrometry analysis.
The aqueous phase was used for liposome flotation assay and protein–lipid
overlay assay.
4
Quantifying CaMKII Isoforms by Western Blot
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As previously described [18] (link), tissue was homogenized using an Omni TH homogenizer, in a (1∶10 w/v) detergent- containing lysis buffer (tissue protein extraction reagent with Halt protease and phosphatase inhibitor cocktail: Thermo Scientific, Waltham, MA). Extracts were centrifuged at 12,000×g for 20 min at 4°C in a Beckman Microfuge 18, and supernatants collected for measurements. Western blots were run using 10 µg of the detergent-solubilized fraction prepared in SDS loading buffer (LI-COR Biosciences, Lincoln, NE). The sample was separated on a 10% NuPAGE Novex Bis-Tris Midi Gel (Life technologies). Proteins were transferred to nitrocellulose membrane using a dry blotting system (iBlot Life Technologies). Blots were probed using reagents and manufacturer recommendations for Odyssey Infrared imaging system (LI-COR Biosciences). Blots were probed for the following primary antibodies: mouse anti-CaMKIIα (1∶10,000, Abcam, Cambridge, United Kingdom, Cat no. ab2725); mouse anti-CaMKIIβ (1∶10,000, LifeSpan Biosciences, Cat no. LS-C21191). Mouse anti-β-actin (1∶10,000, Cell Signaling, Danvers, MA, Cat no. 37005) was used as a loading control. Blots were visualized and analyzed on the Odyssey Infrared imaging system (LI-COR Biosciences).
5
Investigating Lyophilized CSP7 Stability
6
Chondrogenic Gene Expression Analysis
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One week, 2 and 4 weeks post-implantation, a cylindrical tissue sample (3.5-mm in diameter and 1.2-mm in depth) was harvested from the defect site for further detection (n = 4 knees per group for each time point). Chondrogenic differentiation gene expression was analyzed using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) as previously reported (Sun et al., 2018 (link)). Briefly, four independent cylindrical tissue specimens were snap-frozen in liquid nitrogen and then pulverized by a mortar. Total RNA was extracted using a standard TRIzol (Invitrogen, United States) procedure and quantified by a Nucleic Acid and Protein Analyzer (Microfuge18; Beckman-Coulter), followed by cDNA synthesis using a ReverTra Ace® qPCR RT Kit (FSQ-201; TOYOBO). The specific gene primers designed for qPCR are listed in Table 2 , and the experiment was performed using the StepOne TM Real-Time PCR System (Applied Biosystems). The relative gene expression was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and presented as the fold-change relative to the negative control group using the 2ΔΔCt method.
7
DNA Quantification and Electrophoresis
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Centrifugation was performed with a Beckman Coulter Allegra X-15R or Beckman Coulter Microfuge 18. Aqueous solutions of DNA were quantified spectrophotometrically with a Thermo Scientific Nanodrop One. Electrophoresis was performed with an Invitrogen E-GEL Power Snap device using commercial gels (E-Gel EX, 4% agarose) according to manufacturer guidelines. Denaturing electrophoresis was performed using Novex™ TBE-Urea Gels (15%, Invitrogen, EC68855BOX) prepacked gels and Novex™ TBE-Urea Sample Buffer (2X, Invitrogen, LC6876) according to manufacturer guidelines. Completed gels were stained with SYBR™ Gold Nucleic Acid Gel Stain (Invitrogen, S11494) according to manufacturer directions. Oligonucleotide sequences are provided in the Supplementary Information file.
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