The influence of carboydrate exposure on protease activity of B. salamandrivorans zoospores was assessed. Therefore, zoospores were harvested from 175 cm2 cell culture flasks by replacing the TGhL broth with distilled water, which was filtered using a sterile mesh filter with pore size 10 µm (Pluristrainer, PluriSelect). A pool containing approximately 5 × 107 zoospores/ ml was obtained. 200 µl of the spore suspension (107 spores) was added to eppendorfs containing 200 µl H2O (H2O; n = 3), 200 µl 100 mM D-Glucose (Glc; n = 3), 200 µl 100 mM D-mannose (Man; n = 3), 200 µl 100 mM D-galactose (Gal; n = 3), or as a control, 200 µl H2O containing protease inhibitor mix (P8215, Sigma-Aldrich) (PI; n = 3). After 1.5 h at 15 °C, the zoospores were centrifuged for 5 min at 4.000 × g at 15 °C and the supernatant was collected. Protease activity in the supernatant was analyzed using the Pierce Fluorescent Protease Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Three independent repeats of the experiment were performed (biological replicates).
P8215
Manufactured by Merck Group
The P8215 is a laboratory equipment product manufactured by Merck Group. It is designed to perform essential functions in a laboratory setting. The core purpose of this product is to assist in the execution of various scientific experiments and procedures. No further details or interpretations are provided regarding its intended use.
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Lab products found in correlation
Pierce fluorescent protease assay kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti myc sc789, by Santa Cruz Biotechnology (1 mentions)
Anti ha agarose, by Merck Group (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Fastprep 24 homogenizer, by MP Biomedicals (1 mentions)
B per reagent, by Thermo Fisher Scientific (1 mentions)
Anti lexa, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
P0044, by Merck Group (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Lgg sepharose, by GE Healthcare (1 mentions)
Anti gfp, by Roche (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
S8830, by Merck Group (1 mentions)
12 protocols using p8215
1
Carbohydrate Effects on Protease Activity
2
Affinity-based Protein Extraction Protocol
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Strains were grown in selective medium with 2% glucose for experiments with genes expressed from their native promoter, whereas strains were grown overnight in selective medium containing 2% raffinose to logarithmic phase, diluted in the same selective medium containing 2% galactose and incubated at 30°C for 4 hours for experiments with genes expressed from the GAL promoter. Whole cell extracts were prepared by bead beating using a FastPrep-24 homogenizer (MP Biomedicals) in extraction buffer (40mM Hepes, pH7.5, 350mM NaCl, 0.1% Tween, 10% glycerol, protease inhibitors (P8215, Sigma), 1mM DTT, 1mM PMSF). An equal concentration of protein extracts were incubated with anti-HA agarose (A2095, Sigma) at 4°C overnight. The unbound extract was removed following washes in Tris-buffered saline with Tween-20 (0.1%) (TBST) three times, and the immunoprecipitated proteins were eluted in 2X Laemmli buffer or 10 mM Glutathione 50 mM Tris pH8, respectively. Rabbit anti-Myc (sc789, Santa Cruz Inc), mouse anti-Flag (M2, Sigma) and rabbit anti-HA (H6906, Sigma) antibodies were used in Western blot analysis.
3
Optimized Bacterial Protein Extraction
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Briefly, biomass amount equivalent to 1 mL of 1 OD E. coli was collected in an Eppendorf tube and solubilized in a suspension solution consisting of 200 µL B-PER reagent (Thermo Scientific) and 200 µL TEAB buffer (50 mM triethylammonium bicarbonate (TEAB), 1% (w/w) NaDOC, adjusted to pH 8.0) including 0.2 µL protease inhibitor (P8215, Sigma Aldrich). Further, 0.1 g of glass beads (acid, washed, approx. 100 µm diameter) were added and cells were disrupted using three cycles of bead beating on a vortex for 30 s followed by cooling on ice for 30 s in-between cycles. In the following, a freeze/thaw step was performed by freezing the suspension at –80°C for 15 min and thawing under shaking at elevated temperature using an incubator. The cell debris was further pelleted by centrifugation using a bench top centrifuge at max speed, under cooling for 10 min. The supernatant was transferred to a new Eppendorf tube and kept at 4°C until further processed. Protein was precipitated by adding one volume of TCA ( Sigma Aldrich) to four volumes of supernatant. The solution was incubated at 4°C for 10 min and further pelleted at 14,000 g for 10 min. The obtained protein precipitate was washed twice using 250 µL ice cold acetone.
4
Western Blot and Immunoprecipitation Protocols
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For Western blot analysis, yeast cells (O.D600 = 1.5) were collected by centrifugation at 3000 rpm in a table-top centrifuge for 5 min. The cell pellets were resuspended in 100 μl of SDS-buffer (50 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 5 min. After the lysates were cleared by centrifugation at 12,000 rpm for 10 min, soluble proteins were resolved by SDS–PAGE and transferred to PVDF membrane (Millipore). The membranes were incubated with appropriate antibodies (anti-HA, anti-LexA, anti -GFP and anti-TAP antibodies, Santa Cruz) in TBST buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and proteins were detected by the enhanced chemiluminescence (ECL) system. For IP, yeast cells were disrupted by vortexing with acid-washed glass beads in ice cold NP40 buffer (1% NP40, 150 mM NaCl, 50 mM Tris–HCl, pH 8.0) containing protease and phosphatase inhibitors. The cell lysates were incubated with appropriate antibodies at 4 °C for 3 h and further incubated with protein A/G-conjugated agarose beads at 4 °C for 1 h. The precipitated agarose beads were washed three times with ice cold NP40 buffer containing protease and phosphatase inhibitor cocktails (Sigma P8215 and Sigma P0044, respectively) and boiled in 50 μl of SDS–PAGE buffer. The resulting proteins were analyzed by Western blot using appropriate antibodies.
5
Yeast Cell Lysis and Fractionation
6
Protein Extraction from Transgenic Strains
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Strains carrying either the Htt-Q97-GFP or the FFL-Luciferase-GFP reporter were cultured as described above, washed in 10 mM, Tris 1 mM EDTA, pH 8 (TE), and pelleted before freezing at –80°C. The best quality extracts were prepared from samples that had been stored at –80°C for less than 1 wk. After thawing on ice, ∼100 µl of glass beads and 100 µl of extraction buffer (30 mM Tris, pH 8.5, 5 mM EDTA, 3 mM DTT, 5% glycerol plus 2.5 µl of 40 mM PMSF, and 2.5 µl of PIC; Sigma, P8215) was added to pellets. Cells were disrupted with 7 × 30 s Qiagen Tissue Lyser LT cycles alternating with 1 min on ice. The mixture was centrifuged at 2000 rpm for 1 min and the beads and pellet were discarded. The supernatant protein concentration was determined using the Bradford Protein Assay (Bio-Rad) using a bovine serum albumin (BSA) standard curve.
7
Mild CENP-A overexpression analysis
8
MYC Protein Co-immunoprecipitation Protocol
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To prepare WCEs, cells were collected, rinsed in ice-cold PBS with1 mM PMSF, and frozen on dry ice. Lysis was performed in 125 μl ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% Triton X-100, 1 mM DTT, and 10% glycerol) with inhibitors (complete EDTA free protease inhibitor, 40694200; Roche, and 1 mM PMSF) using a bead beater (Cryoprep). After addition of 275 μl ice-cold lysis buffer with protease inhibitors, the extracts were clarified by two successive rounds of centrifugation. Samples were incubated for 1 h at 4°C with anti-MYC magnetic beads (TA150044; Origene). Beads were washed five times with lysis buffer without inhibitors. Proteins were eluted with hot Laemmli buffer at 95°C for 5 min. For reverse co-immunoprecipitation, lysis was performed in ice-cold buffer (50 mM Hepes, pH 7.6, 75 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, 10 mM Sodium butyrate, and 10% glycerol) with inhibitors (1 mM PMSF, 1 mM NaVa, 10 mM β-glycerophosphate, and 1X protease inhibitor [P8215; Sigma-Aldrich]). Samples for each strain were distributed to two tubes. One was treated with 2.5 μg of RNase A (R6513; Sigma-Aldrich) for 20 min on ice. Samples were immunoprecipitated for 1 h at 4°C with anti-FLAG antibody (F1804; Sigma-Aldrich) coupled to μMACS protein G (130-071-101; Miltenyi Biotec).
9
Mitochondrial Inner Membrane Isolation
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Wild type and the Tim17AA mutant strain of Saccharomyces cerevisiae were used for the electrophysiological experiments. Cells were cultivated at 30°C on standard SD (synthetic dextrose minimal medium) with 2% glucose in the absence of histidine for 24 h. Mitochondria were isolated from logarithmically growing cells as described previously (Lohret et al., 1997 (link)). Homogenization buffer was 0.6 M sorbitol, 10 mM Tris, 1 mM EDTA, 0.2% bovine serum albumin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4, containing protease inhibitor mixture (P 8215; Sigma-Aldrich). Mitoplasts were prepared from isolated mitochondria by the French press method (Ohba and Schatz, 1987 (link)), and the inner membranes were further purified according to Mannella (1982) (link). Membrane purity was routinely assessed, and cross-contamination was typically <10%. Inner membranes were reconstituted into giant proteoliposomes by dehydration–rehydration as described previously (Lohret et al., 1997 (link); Muro et al., 2003 (link)) using soybean l -phosphatidylcholine (type IV-S; Sigma-Aldrich).
10
Mushroom Protein Extraction and Purification
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Frozen mushroom pulp was defrosted at 4 °C and suspended in a 125 mM sodium citrate buffer, pH 5.3, containing 25 mM sodium l -ascorbate, 20 mM l -lysine, 50 mM l -proline. Again, PSMF and benzamidine hydrochloride pre-solved in DMSO were added (final concentration 1 mM), as well as two protease inhibitor cocktails (P8215 0.1% (v/v) and S8830 1 tab/L). The suspension was stirred for 15 min and subsequently centrifuged (14,500 rpm at 4 °C over 10 min). The pellet and the highly viscose triton-phase, covering the pellet, were discarded. The supernatant was adjusted to 20% (113 g/L) ammonium sulfate saturation and centrifuged (14,500 rpm at 4 °C over 10 min). The occurring pellet was discarded and PEG-4000 was dissolved to a concentration of 15% (m/v) in the supernatant at 4 °C. After further centrifugation (14,500 rpm, 4 °C, 10 min) the generated PEG phase (upper phase) containing the non-target proteins was discarded. This step was followed by the subsequent addition of another 5% (m/v) of PEG-4000 and centrifugation. This last step was repeated twice. During the extraction/purification process every 45 min a protease inhibitor mixture containing PMSF, benzamidine hydrochloride and the two cocktails P8215/S8830 (Sigma–Aldrich) pre-dissolved in DMSO was added.
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