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Immobilon p

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Immobilon-P is a microporous polyvinylidene difluoride (PVDF) membrane designed for protein transfer and immobilization. It provides high protein-binding capacity and efficient sample transfer during Western blotting and other protein analysis techniques.

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Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (2 mentions) Anti flag m2 affinity agarose beads, by Merck Group (1 mentions) Cox 2 activity assay kit, by Cayman Chemical (1 mentions) High pure rna isolation kit, by Roche (1 mentions) Puromycin, by InvivoGen (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Phosstop, by Roche (1 mentions) P8340, by Roche (1 mentions) Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions) Nbp1 06712, by Novus Biologicals (1 mentions) Anti flag antibody, by Merck Group (1 mentions) Chemidoc imager, by Bio-Rad (1 mentions) Np0323box, by Thermo Fisher Scientific (1 mentions) G8795, by Merck Group (1 mentions) Hybond p, by Cytiva (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Radioimmunoprecipitation assay (ripa), by CWBIO (1 mentions) Fusion fx7, by Vilber (1 mentions)

10 protocols using immobilon p

1

Hemolymph Collection and Western Blot Analysis in Drosophila

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Western blots were performed as previously described65 (link). Haemolymph was collected from 4-day-old male adults. In brief, each fly was cut a small opening at the abdomen (avoid damaging the internal organs, e.g., intestine, etc) using fine-forceps or scissors. The flies were placed into in a 0.5 mL tube (20 flies/tube, small holes were pricked at the bottom) that was placed on top of a 1.5 mL tube containing 5 µl 2× SDS loading buffer. The 1.5 mL tubes were centrifuged at 1 × 104 rpm for 5 min at 20 °C. The total protein was separated on 10% Bis-Tris Protein Gels and transferred onto PVDF membranes (pore size 0.45 μm, Immobilon®-P, Thermoscientific) using constant current 200 mA for 90 min. The membranes were blocked with 5% milk in 1xTBST for 2 hours at room temperature. Incubation with primary antibodies was performed at 4 °C overnight with shaking. Incubation with secondary antibodies was performed at room temperature for 2 hours. The following antibodies were used: rabbit anti-Hh (1:5000), HRP conjugated anti-rabbit (1:5000, GE). The membranes were developed with the Azure 600 imaging system. The protein levels were quantified using Fiji software and normalized against Coomassie Brilliant Blue (CBB) stained total protein on the membranes.
Zhao Y., Khallaf M.A., Johansson E., Dzaki N., Bhat S., Alfredsson J., Duan J., Hansson B.S., Knaden M, & Alenius M. (2022). Hedgehog-mediated gut-taste neuron axis controls sweet perception in Drosophila. Nature Communications, 13, 7810.
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2

Flag-tagged Protein Immunoprecipitation

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Prior to immunoprecipitation, anti-Flag M2 affinity agarose beads (20 μL bead slurry, Sigma-Aldrich) were washed twice with cold TBS (Tris Buffered Saline, 500 μL; 20 mM Tris-Cl at pH 8.0, 150 mM NaCl). Lysates containing either wild type or mutant HDAC-Flag expressed proteins (1 mg total protein) were incubated with prewashed anti-Flag M2 agarose beads at 4°C overnight with rotation. After immunoprecipitation, beads were washed three times with lysis buffer (1 mL) containing high salt (500 mM NaCl). For the LSD1 binding assay, bound proteins were eluted with SDS buffer (20 μL; 100 mM Tris-Cl at pH 6.8, 4% SDS, 20% glycerol, 0.008% bromophenol blue) by boiling at 95°C for 5 min. For the p53 binding assay, bound proteins were eluted with TBS containing 3xFlag peptide (APEXBIO; 40 μL; 0.25 mg mL−1 in TBS) for 30 min at 4°C. The eluted proteins were mixed with SDS loading dye (10 μL; SDS buffer containing 10% v/v β-mercaptoethanol) and boiled at 95°C for 2 min. As controls, lysates without immunoprecipitation (50 μg) were denatured with SDS loading dye (5 μL) by boiling at 95°C for 2 min. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membrane (Immobilon P, Fischer Scientific), and immunoblotted with monoclonal FLAG® M2 (F3165, Sigma), LSD1 (L4418, Sigma) or p53 (sc-126, SantaCruz Biotechnology) antibodies.
Gomes I.D, & Pflum M.K. (2019). Optimal substrate trapping mutants to discover substrates of HDAC1. Chembiochem : a European journal of chemical biology, 20(11), 1444-1449.
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3

Evaluating the Effects of Vitamin D Analogs and COX-2 Inhibitors

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1α,25(OH)2D3, and the antibiotic G418 were from Sigma-Aldrich (St. Louis, MO, USA). Puromycin was provided by Invivogen (San Diego, CA, USA). The antibodies used were rat monoclonal anti-VDR (Affinity Bioreagents, Golden, CO, USA); mouse monoclonal anti-COX-2, anti-mouse and anti-rat horseradish peroxidase–conjugated secondary antibody (Santa Cruz, CA, USA). Roche Applied Science (Indianapolis, IN, USA) provided high Pure RNA Isolation Kit. Immobilon P (polyvinylidene difluoride; PVDF) membranes were from Thermo Scientific (Rockford, IL, USA); PCR primers for mouse Gapdh, Cox-2, EP1, EP2, EP3 and EP4 were synthetized by Invitrogen (Thermo Scientific Inc., Rockford, IL, USA). Celecoxib (Santa Cruz, CA, USA). COX-2 Activity Assay kit (Cayman N° 760151) was from Cayman Chemical Company (Michigan, USA).
Tapia C., Zamarreño F., Salvador G.A., Casali C.I., Viso J., Fernandez M.D., White J.H, & González-Pardo V. (2020). Down-regulation of COX-2 activity by 1α,25(OH)2D3 is VDR dependent in endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor. Heliyon, 6(10), e05149.
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4

Western Blot of Microdissected DG Proteins

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Micro-dissected DG tissues were homogenized on ice in RIPA buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM sodium fluoride, Protease Inhibitor Cocktail [Sigma-Aldrich]) using disposable pellet pestles. Sample concentration was determined using the Bradford method. Samples were diluted in RIPA buffer to a final concentration of 2 ug/uL in 1× loading buffer (50 mM Tris-CL pH6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 5 mM DTT, 0.02% bromophenol blue). Samples were resolved on an 8% Tris-glycine SDS-PAGE gel and blotted by wet transfer onto PVDF membranes (Immobilon-P; Thermo Fisher Scientific Inc.). Membranes were washed with TBS-T (20 mM Tris base, 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk in TBS-T, and incubated overnight at 4 °C with primary antibodies. Membranes were washed and incubated with secondary antibodies for 2 hr at RT. Band signals were detected by chemiluminescence using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific), exposed on film and developed for optimal visualization. Band intensity was measured using the ImageJ software (http://rsbweb.nih.gov/ij). Antibody concentrations can be found in Table S3.
Bouchard-Cannon P., Lowden C., Trinh D, & Cheng H.Y. (2018). Dexras1 is a homeostatic regulator of exercise-dependent proliferation and cell survival in the hippocampal neurogenic niche. Scientific Reports, 8, 5294.
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5

Immunoprecipitation and Western Blot Analysis

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HEK 293 cells and kidneys from fed animals were homogenized in lysis buffer (Tris 50 mM pH 7.9, 1% triton X-100, 0.1% Tween20, 150 mM NaCl, 10% glycerol, 5 mM MgCl2) or in a specific buffer for Co-IP experiments (50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 0.5% Triton X-100, pH 7.28). For Co-IP experiments, lysates were incubated with specific antibodies and IgG, as control, as described65 (link). Co-IP experiments were performed at least three times. Western blot (WB) studies were performed at least in triplicate and representative images were shown. For Co-IP experiments the ratio of IP proteins with respect to the input was 1:50. Blots were quantified by ImageJ. The control was settled as 1 and the fold change was calculated and reported below the panels as the mean ± standard error of the mean (SEM). Animals were perfused with PBS to eliminate blood traces for detection of the GH protein. Lysates were treated with protease inhibitors from Sigma-Aldrich (P8340) and phosphatase inhibitors from Roche (PhosSTOP, 04906837001). Polyvinylidene difluoride (PVDF) membranes were used for Immunoblot (Millipore, US, Immobilon-P, IPVH00010) and ECL western blotting reagent (Thermoscientific, 32106) or Femto (Thermoscientific, 34095) were used for detection.
Iaconis D., Monti M., Renda M., van Koppen A., Tammaro R., Chiaravalli M., Cozzolino F., Pignata P., Crina C., Pucci P., Boletta A., Belcastro V., Giles R.H., Surace E.M., Gallo S., Pende M, & Franco B. (2017). The centrosomal OFD1 protein interacts with the translation machinery and regulates the synthesis of specific targets. Scientific Reports, 7, 1224.
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6

Immunoblotting and Co-immunoprecipitation of ATP10B and CDC50A

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HeLa microsomes were prepared according to [49 (link)] and immunoblotting was performed as previously described [113 (link)]. For total cell lysate investigation, cells were lysed using RIPA buffer (ThermoFisher Scientific, 89900) and DNA excluded by centrifugation (15,000×g for 15 min). Briefly, western blots of typically 20–40 µg of protein were ran on 4–12% Bis/Tris gel (NuPage, Thermo Scientific, NP0323BOX) and transferred to a 0.45 µm PVDF membrane (Immobilon-P, Thermo Scientific, 88518) and probed for ATP10B (Sigma-Aldrich, HPA034574), CDC50A (anti-FLAG antibody; Sigma-Aldrich, F3165). Cell death was assessed using a cleaved caspase 3 antibody (Cell signaling, 9661). All blots were probed for either GAPDH (Sigma-Aldrich, G8795) or β-actin (Sigma-Aldrich, C6198) as a loading control. Detection was performed using HRP-conjugated secondary antibodies (BIOKE, 7074S and 7076S). Detections were performed on a Bio-Rad Chemidoc Imager with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106). For co-immunoprecipitation experiments, PVDF membranes (Hybond P; Amersham Biosciences) were blocked in 5% skimmed milk in PBS and probed with primary antibodies against HA-tag (Covance, MMS-101P) and FLAG-tag (Novus Biologicals, NBP1-06712).
Martin S., Smolders S., Van den Haute C., Heeman B., van Veen S., Crosiers D., Beletchi I., Verstraeten A., Gossye H., Gelders G., Pals P., Hamouda N.N., Engelborghs S., Martin J.J., Eggermont J., De Deyn P.P., Cras P., Baekelandt V., Vangheluwe P, & Van Broeckhoven C. (2020). Mutated ATP10B increases Parkinson’s disease risk by compromising lysosomal glucosylceramide export. Acta Neuropathologica, 139(6), 1001-1024.
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7

Western Blot Analysis of Protein Expression

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The cells were lysed by the addition of RIPA (cat: #CW2333,CWBio,Beijing, China) containing protease inhibitors (cat: #A32961,ThermoFisher Scientific, Waltham, MA, USA).
Proteins were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene fluoride membranes (Immobilon-P,ThermoFisher Scientific). The membrane was blocked at 25℃with 5% fat-free milk in phosphate-buffered saline (PBS) and 0.1% Tween-20 (0.1% PBS-T) for 1 h and then incubated with the appropriate primary antibody [15-LOX-2, 1:200; caspase-3, 1:1000; cyclophilinB (CypB), 1:1000; β-tubulin, 1:1000] (Table2) overnight at 4°C, followed by incubation with the appropriate secondary antibody (anti-rabbit or anti-mouse IgG HRP-linked, 1:10000) for 1 h at room temperature. Immunoreactive proteins were detected using the VilberLourmat imaging system (Fusion Fx7,VilberLourmat,Marne-la-ValléeCedex 3, France).
Huang R., Li T.L., Xi Y., Wen H.W., Zhou X.Z., Liao Y.L., You J.Y., Yu C.Y., Xu P., Wang Y.W., Wen D.W., Xia T.X., Yang H., Chen Y.C., Xu L.X., Zhong X.Z., Li X.L., Zhou C.Z., & Xu Z.X. (2021). 15-Lipoxygenase-2 Deficiency Induces a Dysfunction in Macrophages That Can Be Restored by Salidroside.
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8

Detecting RCAN1-FLAG Protein Expression

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To test for expression of RCAN1-FLAG, RNA was extracted from cells grown to confluence on a 6 cm plate using TRIzol reagent (Invitrogen). cDNA was generated by RT-PCR using Superscript III enzyme (Invitrogen). PCR reactions were carried out using RCAN1-5′-utrF and RCAN1-3′-FLAGR primers. RCAN1-FLAG protein expression levels were assessed by Western blot analysis. Protein was extracted from PC12 clones and run on a 12% SDS-PAGE gel and transferred onto a PVDF membrane (Immobilon-P, Invitrogen). An α-FLAG antibody conjugated to horseradish peroxidase (Sigma) was used to probe the membrane at a dilution of 1 : 1000.
Peiris H., Dubach D., Jessup C.F., Unterweger P., Raghupathi R., Muyderman H., Zanin M.P., Mackenzie K., Pritchard M.A, & Keating D.J. (2014). RCAN1 Regulates Mitochondrial Function and Increases Susceptibility to Oxidative Stress in Mammalian Cells. Oxidative Medicine and Cellular Longevity, 2014, 520316.
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9

Protein Extraction and Analysis from Plant Cells

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Cells were pelleted by centrifugation at 3500 × g for 5 min at 4°C, washed with PBS and resuspended in 50 mM Tris pH 8, SDS 9%, PID, PMSF 0.1 M and NaCl 150 mM. Total protein extracts were obtained by freeze/thaw cycles in liquid nitrogen followed by another mechanical disruption procedure as described above. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Comassie blue, and transferred onto a PVDF membrane (Immobilon ®-P, pore size 0.45 μm) using a power blotting station (Invitrogen™, Thermo Fischer). Immunoblotting analysis was performed with an antibody against photosystem II subunit S (PsbS) at 1:1000 (Agrisera). Anti-rabbit secondary antibody was used at 1:10,000 (Invitrogen). Immunoblots were visualized using IQ800 Control software (ImageQuant 800, Amersham).
Serrano-Pérez E., Romero-Losada A.B., Morales-Pineda M., García-Gómez M.E., Couso I., García-González M, & Romero-Campero F.J. (2022). Transcriptomic and Metabolomic Response to High Light in the Charophyte Alga Klebsormidium nitens. Frontiers in Plant Science, 13, 855243.
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10

Western Blot Analysis with ScanLater Imaging

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Western blotting was carried out with the Molecular Devices ScanLater system (San Jose, CA), as described previously40 (link). Briefly, cells were harvested in RIPA buffer with protease and phosphatase inhibitors and agitated for an hour at 4 °C before protein quantification with a Pierce BCA (bicinchoninic acid) assay. Equal amounts of protein were loaded on precast Bolt 4–12% Bis/Tris gels (Invitrogen) and run at 150 V for 45–60 min in Bolt MOPS(3-(N-morpholino)propanesulfonic acid) buffer before transfer onto a polyvinylidene fluoride membrane (Immobilon-P) in Bolt transfer buffer for 1.5 h at 25 V. Membranes were cut horizontally to enable high quality imaging with multiple antibodies per blot, blocked with 1 × ScanLater buffer, probed with primary antibodies overnight, rinsed with ScanLater wash buffer, and then probed with Eu-labeled (europium labeled) secondary antibodies (goat antimouse: R8205, goat antirabbit: R8204) for 1 h before rinsing membranes and drying them. Dry membranes were imaged with a SpectraMax i3x with a Western blot cartridge (Molecular Devices). Running and transfer buffers were obtained from ThermoFisher Scientific (Waltham, MA), and blots were normalized to loading control, vinculin, and quantified with Licor ImageStudio Lite Version 5.2 software (Lincoln, NE).
Weissenrieder J.S., Weissenkampen J.D., Reed J.L., Green M.V., Zheng C., Neighbors J.D., Liu D.J, & Hohl R.J. (2022). RNAseq reveals extensive metabolic disruptions in the sensitive SF-295 cell line treated with schweinfurthins. Scientific Reports, 12, 359.
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