The SPE cartridges (Oasis MCX) were preconditioned with 2 mL methanol and 2 mL distilled water, sequentially. After being preconditioned, the centrifuged plasma sample was then loaded into the cartridges, previously washed with 2 mL distilled water and 2 mL cyclohexane, and dried under reduced pressure for 5 min. Elution of analytes was carried out with 2 mL of methanol. 100 μL of eluted methanol was filtered with 0.22 μm PVDF microporous membrane (Millipore, Billerica, MA) and introduced into 200 μL vials. Finally, 1 μL of final solution was injected into the GC-MS.
Pvdf microporous membrane
Manufactured by Merck Group
Sourced in United States, Germany
PVDF (polyvinylidene fluoride) microporous membrane is a type of filtration material used in various laboratory and industrial applications. It is composed of a porous polymer structure that allows the passage of fluids and particles while retaining specific components based on their size. The core function of this membrane is to facilitate efficient separation and filtration processes, enabling the isolation or purification of target substances from complex mixtures.
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Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
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Bca protein assay kit, by CoWin Biotech (3 mentions)
Protease inhibitor, by Applygen (3 mentions)
Gapdh, by Abcam (3 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Western light chemiluminescent detection system, by Thermo Fisher Scientific (2 mentions)
Nih imagej software, by Bio-Rad (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Ab4052, by Abcam (2 mentions)
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Ripa lysis buffer, by Applygen (2 mentions)
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Bca detecting kit, by Keygen Biotech (1 mentions)
39 protocols using pvdf microporous membrane
1
Plasma Sample Preparation for GC-MS
2
Western Blot Protein Analysis
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After transfection, cells were harvested and the total protein was extracted from cells using RIPA lysis buffer. The quality of protein was qualified using BCA detecting kit (Keygen, Nanjing, China). Sample was subjected SDS-PAGE (10%) and then transferred to PVDF microporous membrane (Millipore, Boston, MA, USA). Blots were probed by rabbit polyclonal antibodies anti-WTAP (1:1000, 195,380) at 4°C overnight. After that, membranes were incubated with secondary antibodies HRP-goat anti-rabbit (ABclonal) at room temperature. Protein bands were, respectively, detected on chemiluminescence imaging analysis system using Enhanced chemiluminescence (ECL, Millipore, Bredford, USA).
3
Hippocampal Protein Analysis by Western Blot
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Hippocampi were homogenized in RIPA buffer. The homogenates were centrifuged for 15 min at 12,000 g at 4 °C. The quantity of protein in each supernatant was determined using a BCA protein assay kit. Proteins (60 μg) were denatured with sodium dodecyl sulfate (SDS) sample buffer and separated using 10 % SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Bedford, MA, USA), which was then blocked with 5 % skim milk for 1 h at room temperature. The membrane was incubated with primary antibody overnight at 4 °C. The following primary antibodies were used: rabbit polyclonal anti-APP, -iNOS, -COX-2, and -BACE1 and mouse monoclonal anti-GAPDH (1:1000). After adding the anti-rabbit or anti-mouse secondary antibody (1:1000) for 1 h, the protein bands on the membranes were detected with ECL kits (Thermo Fisher Scientific, Rockford, IL, USA). The relative density of the protein bands was scanned by densitometry using Image Lab software (Bio-Rad, Richmond, CA, USA) and quantified by NIH ImageJ software (Bethesda, MD, USA).
4
Investigating miR-373 Regulation in MG-63 Cells
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Standard Western blotting was conducted for protein expression assays from MG-63 cells with miR-373 mimic, inhibitor, and miR-control. Briefly, proteins were isolated with RIPA lysis buffer containing 1 mg of protease inhibitors (Applygen Technologies Inc., Beijing, P.R. China) after 2 days of transfection. The protein content was quantified using Bicinchoninic Acid (BCA) Protein Assay Kit (CoWin Biotech Co., Ltd., Beijing, P.R. China). The following primary antibodies p53 (ab1101), p21 (ab109520), p53 upregulated modulator of apoptosis (Puma; ab9643), B-cell lymphoma-2 associated X (Bax; ab32503), plasminogen activator inhibitor (PAI; ab66705), PI3K (ab86714), AKT (ab8805), Rac1; ab33186), c-Jun N-terminal kinase (JNK; ab124956), and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Abcam (Cambridge, UK). Subsequently, secondary antibodies were marked by horseradish peroxidase for 2 h at 37°C. Samples were then electrotransferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). The bands were visualized by the Odyssey CLx equipment (LI-COR Bioscences, Lincoln, NE, USA).
5
CD147-CD98 Protein Interaction Analysis
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Cell extracts (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a polyvinylidene difluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). The membrane was incubated with primary antibodies against CD147, CD98, integrin, and α-tubulin, according to the manufacturer’s instructions. Horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) were applied to the membrane and detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL). For the pull-down assay, 10 μg of the HAb18 mAb was first immobilized onto AminoLink Plus Coupling Resin (Pierce kit, Lot: 26149). Then, the bait (CD147-ED) and prey proteins (different amount of CD98-ED) were mixed. The protein mixture and controls (CD98-ED only) were then added to the appropriate resin and incubated. After the resin was washed three times with PBS, the eluted samples were subjected to western blotting.
6
Protein Expression Analysis by Western Blot
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Cells were lysed with RIPA cell lysis buffer (Beyotime, Shanghai, China), and the protein quantification was determined by BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Equal quantities of protein were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA). After being blocked with 5% fat-free milk, the membrane was probed with primary antibodies including rabbit anti-MMP-14 (Abcam, ab51074), HAb18 against CD147 (prepared by our laboratory), rabbit anti-CTR1 (Epitomics, 5773-1), rabbit anti-AKT (Cell Signaling Technology, 4685), rabbit anti-phospho(S473)-Akt (Abcam, ab81283). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz, sc-2004 and sc-2005), protein bands on the membrane were visualized using a chemiluminescence kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Mouse antibody against α-tubulin (Abcam, ab80779) or β-actin (Abcam, ab6276) was used to control for differences in protein loading.
7
Western Blot Analysis of Microglial TLR4 and PI3K/Akt Pathway
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Cellular proteins were extracted from the primary microglial cells using RIPA buffer. The protein concentration in the supernatant fluid of the lysate was measured using a BCA kit. Proteins (50 μg) in the cell extracts were denatured with sodium dodecyl sulfate (SDS) sample buffer and separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Bedford, MA, USA), which was then blocked with 5% skim milk for one hour at room temperature. The membrane was incubated with primary antibody overnight at 4°C. The following primary antibodies were used: rabbit monoclonal anti-TLR4, −p-PI3K (p85α), −p-Akt (ser473), −p-FoxO1, −PI3K, −Akt, −FoxO1, and mouse monoclonal anti-GAPDH (1:1000). After adding the anti-rabbit or anti-mouse secondary antibody (1:1000) for 1 hour, the protein bands on the membranes were detected with an enhanced chemiluminescence kit.
8
Protein Isolation and Western Blot Analysis
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Cells were lysed in 1% OG buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% OG, 1 mM EDTA, 10 μg/ml leupeptin, 2 μg/ml aprotinin and 1 mM PMSF). A BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Illinois) was then used to determine the total protein concentration, and equal amounts of protein were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Billerica, MA). The membrane was blocked with 5% non-fat milk and then incubated for 2 h at room temperature with the designated antibody. The Western-Light chemiluminescent detection system (Applied Biosystems, Foster, CA) was used for immunodetection.
9
Western Blot Analysis of Signaling Proteins
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Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore Corporation, MA, USA) for Western blotting. The membrane was incubated at 4°C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, GLUT4 (1∶1000), E-cadherin (1∶200), and β-actin (1∶2000). After the incubation, anti-mouse IgG horseradish peroxidase-conjugate (HRP, 1∶2000, for β-actin) or anti-rabbit IgG HRP (1∶2000, for p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, and GLUT4) was added; and the membrane was incubated for 30 min at room temperature. The antigenic proteins on the membrane were visualized by chemiluminescence by use of a Lumi-LightPLUS Western blotting kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated by using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan).
10
Western Blot Protein Expression Analysis
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Protein expression was analyzed by Western blotting as previously described [39] (link). In brief, cells were lysed in 1% n-octyl-p-D-glucopyrano-side (OG) buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% OG, 1 mM EDTA, 10 g/ml leupeptin, 2 g/ml aprotinin,1 mM PMSF). BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) was employed to determine the total protein density and equal amounts of proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then electransferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated for 2 h at room temperature with the designated antibody. Immunodetection was performed by using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster, CA, USA).
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