Immobilon fl membrane

Manufactured by Merck Group

Sourced in United States, Germany, Sweden, Morocco

Immobilon-FL membrane is a fluorescent protein-compatible polyvinylidene fluoride (PVDF) membrane designed for Western blotting. It provides a high-binding capacity for protein transfer and detection.

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Lab products found in correlation

Odyssey infrared imaging system, by LI COR (21 mentions) Odyssey blocking buffer, by LI COR (16 mentions) Odyssey scanner, by LI COR (9 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions) Odyssey clx imaging system, by LI COR (7 mentions) Odyssey imaging system, by LI COR (6 mentions) Protease inhibitor cocktail, by Merck Group (6 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (6 mentions) Odyssey imager, by LI COR (5 mentions) Gapdh, by Cell Signaling Technology (5 mentions) Laemmli sample buffer, by Bio-Rad (5 mentions) Mouse anti β actin, by Merck Group (5 mentions) Protease inhibitor cocktail, by Roche (5 mentions) Sample buffer, by Bio-Rad (4 mentions) Chemidoc mp system, by Bio-Rad (4 mentions) Irdye 800cw goat anti mouse, by LI COR (4 mentions) Cell lysis buffer, by Cell Signaling Technology (4 mentions) Blocking buffer, by LI COR (4 mentions) Ripa buffer, by Merck Group (4 mentions) Ab6276, by Abcam (3 mentions)

150 protocols using immobilon fl membrane

Generating Polyclonal DGN Antibodies

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Polyclonal DGN antibody Sheep173 was made by immunizing a sheep with a His-tagged rabbit DGN protein (amino acid 1–315 of α-DG) grown in a stable mammalian cell line [HEK293 cells; American Type Culture Collection (ATCC)] and purified by Talon beads (Takara Bio USA, Inc.). The anti-DGN antibodies in the sheep serum were affinity purified by affinity strips containing rabbit DGN purified from E. coli and transferred to a polyvinylidene difluoride membrane (Immobilon FL-Membrane; Millipore) to enrich antibodies to α-DGN lacking glycans and, thus, exclude any carbohydrate-specific antibody epitopes. Polyclonal α-DG antibody Sheep174 targeting the mucin region of α-DG (amino acid 316–485 of α-DG) was made by immunizing a sheep with a His-tagged protein grown in a stable mammalian cell line (HEK293 cells; ATCC) and purified by Talon beads (Takara Bio USA, Inc.). The anti-α-DG antibodies to the mucin region were affinity purified by affinity strips containing GST-mucin α-DG purified from E. coli and transferred to a polyvinylidene difluoride membrane (Immobilon FL-Membrane; Millipore). Using E. coli protein of the mucin α-DG region for affinity purification ensured we enriched for antibodies to α-DG lacking glycans in the mucin region and excluded any carbohydrate-specific antibody epitopes.

de Greef J.C., Slütter B., Anderson M.E., Hamlyn R., O’Campo Landa R., McNutt E.J., Hara Y., Pewe L.L., Venzke D., Matsumura K., Saito F., Harty J.T, & Campbell K.P. (2019). Protective role for the N-terminal domain of α-dystroglycan in Influenza A virus proliferation. Proceedings of the National Academy of Sciences of the United States of America, 116(23), 11396-11401.

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Western Blot Protein Detection Protocol

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The protein samples were resolved in 10% SDS-PAGE gel electrophoresis before being transferred to an Immobilon FL membrane (Millipore.) Membranes were blocked in Tris buffered saline solution containing 1% Tween-20 and 0.25% bovine skin gelatin. The membranes were incubated with primary antibodies overnight at in the blocking solution. After removing the primary antibody and washing the membrane in the blocking solution, the secondary antibodies were applied in the blocking solution for 1 hour. Membranes were washed again in the blocking solution and imaged on a Typhoon 9410 imager (GE) after washing with PBS.

Murphy D., Cieply B., Carstens R., Ramamurthy V, & Stoilov P. (2016). The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina. PLoS Genetics, 12(8), e1006256.

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Giardia Parasite Protein Extraction

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Giardia parasites were iced for 30 min then centrifuged at 700 x g for 7 min and washed twice in 1X HBS supplemented with HALT protease inhibitor (Pierce) and phenylmethylsulfonyl fluoride (PMSF). The cells were resuspended in 300 µl of lysis buffer contains 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 7.5% glycerol, 0.25 mM CaCl₂, 0.25 mM ATP, 0.5 mM Dithiotheitol, 0.5 mM PMSF (Phenylmethylsulfonyl flroride), 0.1% Trition X-100 and Halt protease inhibitors (Pierce). The sample was pelleted at 700 x g for 7 min, the supernatant was mixed with 2X sample buffer (Bio-Rad) and boiled at 98°C for 5 min. Protein samples were separated using sodium dodecyl sulfate (SDS) polyacriamide gel electrophoresis. Protein was transferred to Immobilon-FL membrane (Millipore). To detect tubulin, a mouse monoclonal anti-acetylated tubulin clone 6-11B-1 antibody (IgG2b; product T 6793; Sigma-Aldrich) were used at 1:2,500 and secondary anti-mouse isotype-specific antibody conjugated with Alexa 488 (anti-IgG2b) were used at 1:2,500. To detect CWP1, Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was used at 1:2,000. Multiplex immunoblots were imaged using a Chemidoc MP system (Bio-Rad).

Shih H.W., Alas G.C, & Paredez A.R. (2023). Encystation stimuli sensing mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia. Research Square.

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Western Blotting of P2Y12 Receptor

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Protein lysates in lysis buffer were mixed with 2× Laemmli buffer and heated to 95 °C for 5 min before being loaded onto a Precast Mini-Protean^® TGX™ gels (456-9034, Biorad, Solna, Sweden). After separation, proteins were transferred to an Immobilon^®-Fl membrane (IPFL0010, Millipore, Solna, Sweden). The membrane was blocked using the Intercept^® (Tris Buffered Saline) blocking buffer (927-60001, Li-Cor, Bad Homburg, Germany) diluted 1:4 in PBS, and sequentially incubated with P2Y12 antibodies and goat-anti-rabbit Alexa 680 (A21088, ThermoFisher, Uppsala, Sweden) PBS + 0.1% Tween^®20 or blocking buffer with 0.1% Tween^®20 and 0.01% SDS, respectively. Beta-actin antibodies were used as loading control (ab6276, Abcam, Cambridge, UK) and incubated with IRDye 800CW Goat anti-mouse (Li-Cor, Bad Homburg, Germany,). All incubations were carried out at room temperature for 1 h or overnight at 4 °C. The membranes were scanned using an Odyssey scanner (Li-Cor, Bad Homburg, Germany).

Pavlović N., Kopsida M., Gerwins P, & Heindryckx F. (2020). Inhibiting P2Y12 in Macrophages Induces Endoplasmic Reticulum Stress and Promotes an Anti-Tumoral Phenotype. International Journal of Molecular Sciences, 21(21), 8177.

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Western Blot Analysis of Protein Markers

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Gels were transferred to methanol-activated Immobilon-FL membrane (Millipore) in the transfer buffer (25 mM Tris-base,192 mM glycine, 10 % methanol) at 75 V for 2 hours. Proteins were probed with anti-alpha tubulin (1:1000; Sigma #T6074), anti-APT1 (1:500; Millipore; #MABS166), anti-APT2 (1:500; Thermo; #PA-527653), anti-Pan Ras (1:1000; Millipore; #05–516), anti-Scrib (1:1000; Millipore; #MAB1820), anti-MAP6 (1:1000; anti-STOP; Cell Signaling Technology; #4265), or anti-ABHD17A (1:1000; Aviva Systems Biology; #ARP67573_P050) for 16 hours at 4°C and then incubated with secondary anti-mouse or anti-rabbit horseradish peroxidase (HRP;1:1000; Thermo; #32430) for 1 hour at room temperature. Blots were developed by West Pico Chemiluminescent Substrate (Thermo) and then the chemiluminescence was detected by Azure c600 imager (Azure Biosystems).

Won S.J, & Martin B.R. (2018). Temporal profiling establishes a dynamic S-palmitoylation cycle. ACS chemical biology, 13(6), 1560-1568.

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Western Blot Analysis of Protein

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Fifty micrograms of cell lysate was electrophoresed on a denaturing reducing 4–12% polyacrylamide gel. Protein was transferred to Immobilon-FL membrane (Millipore), blocked for 2 h at RT in Odyssey blocking buffer (LI-COR) prior to incubation with ab84058 (abcam) at 1:500 dilution overnight at 4 °C. Signal was detected by anti-rabbit IRDye-680LT secondary antibody (LI-COR) on an Odyssey infrared detection system (LI-COR). See Supplementary Fig. 9 for uncropped western blot.

Keightley M.C., Carradice D.P., Layton J.E., Pase L., Bertrand J.Y., Wittig J.G., Dakic A., Badrock A.P., Cole N.J., Traver D., Nutt S.L., McCoey J., Buckle A.M., Heath J.K, & Lieschke G.J. (2017). The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger. Nature Communications, 8, 14911.

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Western Blot Protein Analysis

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Western blot analyses were performed as described previously (Elliott et al., 2018 ; Vo et al., 2013 (link); Zhong et al., 2012 (link)). Briefly, protein samples (30–35 µg proteins) were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, Massachusetts). After blocking, the membranes were incubated with appropriate dilutions of specific primary antibodies (1:1000 for p-AKT, AKT, p-EGFR, EGFR,; 1:500 for Giα2; 1:5000 for β-actin: 1:3000 for α-tubulin) overnight at 4°C. After washing, the blots were incubated with appropriate secondary antibodies and developed in ECL mixture. The blots were then exposed to an X-ray film and visualized by autoradiography. The density of specific protein bands was determined by ImageJ software (NIH, Bethesda, MD) and normalized using density of β-actin or α-tubulin bands which were used as loading controls. For RGS2, protein samples (40 µg) were loaded on 15% SDS-PAGE gel, electrophoresed, and transferred to an Immobilon-FL membrane (Millipore). Primary antibodies were used to identify RGS2, HA tag and loading control β-actin. IRdye700- or IRdye800-labeled secondary antibodies were used for protein band detection. The images were captured with a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) at wavelengths of 700 or 800 nm.

Caggia S., Chunduri H., Millena A.C., Perkins J.N., Venugopal S.V., Vo B.T., Li C., Tu Y, & Khan S.A. (2018). Novel role of Giα2 in cell migration: Downstream of PI3-kinase/AKT and Rac1 in prostate cancer cells. Journal of cellular physiology, 234(1), 802-815.

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Immunoblotting for Protein Detection

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Cells were lysed in RIPA buffer and protein concentrations were determined by Bradford assay (Biorad). Proteins were separated by SDS–PAGE electrophoresis with a 4–12% Bis-Tris protein gel (Thermo Scientific) and then transferred to an Immobilon-FL membrane (Millipore). Blots were probed with primary antibodies, rabbit α-HA (1:1000, Abcam #ab9110), rabbit α-FKBP (1:1000, Abcam #ab2918), rabbit α-FRB (1:1000, Crabtree lab) and mouse α-GAPDH (1:2000, Santa Cruz #sc32233) followed by fluorescence-conjugated secondary antibodies (Li-Cor) and bands were detected using an Odyssey CLX imaging system (Li-Cor).

Braun S.M., Kirkland J.G., Chory E.J., Husmann D., Calarco J.P, & Crabtree G.R. (2017). Rapid and reversible epigenome editing by endogenous chromatin regulators. Nature Communications, 8, 560.

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Protein Quantification and Western Blot

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Bicinchoninic acid (BCA) assay (Pierce, 23225) or 660 nm assay (Pierce, 22662) was used to determine protein concentration according to the manufacturer’s instructions. NuPAGE 4–12% gradient Bis-Tris precast gels (Life Technologies, NPO336) were used for SDS-PAGE, followed by transfer onto methanol-activated polyvinylidine fluoride (PVDF) membrane (Immobilon-FL membrane, pore size 0.45 μm; Millipore, IPFL00010). Membrane was blocked, then sequentially labelled with primary and secondary antibodies. Fluorescence detected by scanning with a LI-COR Odyssey scanner and Image Studio analysis software (LI-COR Biosciences).

Daly J.L., Danson C.M., Lewis P.A., Zhao L., Riccardo S., Di Filippo L., Cacchiarelli D., Lee D., Cross S.J., Heesom K.J., Xiong W.C., Ballabio A., Edgar J.R, & Cullen P.J. (2023). Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health. Nature Communications, 14, 3086.

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Quantifying Gli3 Protein Levels in Mutant Embryos

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Protein was extracted from the dorsal telencephalon of E12.5 wild-type and Inpp5e^Δ/Δ embryos (n = 4 samples per genotype) as described previously (Magnani et al., 2010 (link)). 10 μg protein lysates were subjected to gel electrophoresis on a 3–8% NuPAGE Tris-Acetate gel (Life Technologies), and protein was transferred to a Immobilon-FL membrane (Millipore), which was incubated with goat anti-h/m Gli3 (1:500, R and D Systems #AF3690) and mouse anti-β-Actin antibody (1:15000, Abcam #ab6276). After incubating with donkey anti-goat IgG IRDye680RD (1:15000, LI-COR Biosciences) and donkey anti-mouse IgG IRDye800CW secondary antibodies (1:15000, Life Technologies), signal was detected using LI-COR’s Odyssey Infrared Imaging System with Odyssey Software. Values for protein signal intensity were obtained using Image Studio Lite Version4.0. Gli3 repressor and full-length protein levels and the Gli3 repressor/full length were compared between wild-type and mutant tissue using an unpaired t-test.

Hasenpusch-Theil K., Laclef C., Colligan M., Fitzgerald E., Howe K., Carroll E., Abrams S.R., Reiter J.F., Schneider-Maunoury S, & Theil T. (2020). A transient role of the ciliary gene Inpp5e in controlling direct versus indirect neurogenesis in cortical development. eLife, 9, e58162.

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