Polyisobutylmethacrylate

Manufactured by Merck Group

Sourced in Germany

Polyisobutylmethacrylate is a synthetic polymer with a primary function as a laboratory equipment component. It is a clear, colorless, and thermoplastic material that exhibits chemical and thermal stability. The material is often used in various laboratory applications that require durable and inert components.

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5 protocols using polyisobutylmethacrylate

Gb3Cer Detection in Cell Extracts

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For the detection of Gb₃Cer, two GSL-aliquots of Lovo and HCT116 control- and Genz-treated cell extracts, each corresponding to 0.2 mg protein were loaded on a TLC plate (Merck, Darmstadt, Germany). The TLC plate was divided into two parts. One was stained with orcinol reagent as described above to visualize all GSLs. The second plate was fixed with 5% polyisobutylmethacrylate (Sigma, Munich, Germany) in chloroform which was 1:10 diluted in n-hexane. After the organic solvent was evaporated, the plate was blocked with 1% BSA in PBS for 30 min. Blocking solution was removed and the plate subsequently covered with polyclonal chicken anti-Gb₃Cer antibody 1:200 diluted in 1% BSA in PBS at 4 °C overnight. The plate was washed five times with PBS/0.05% Tween-20 and incubated with secondary 1:500 diluted alkaline phosphatase linked donkey anti-chicken IgY antibodies (Dianova, Hamburg, Germany) in 1% BSA/PBS for 4 h at RT. After washing for five times, positive bands were detected using Sigma Fast BCIP (Sigma, Munich, Germany).

Jennemann R., Volz M., Bestvater F., Schmidt C., Richter K., Kaden S., Müthing J., Gröne H.J, & Sandhoff R. (2021). Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo. International Journal of Molecular Sciences, 22(19), 10539.

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Purification and Characterization of Compounds

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Methanol (p.a.) was purchased from VWR International AB. Ethanol (>99.5%) was purchased from Solveco AB. Toluene (HPLC grade, >99.8%) was purchased from RCI Labscan. Chloroform (≥99.8%), lithium chloride (≥99%), potassium hydroxide, sulfuric acid (95–97%), acetic acid glacial (100%), dichloromethane (anhydrous, stabilized with amylene, ≥99.8%), acetic anhydride (p.a., >99.5%), pyridine (>99.5%), anisaldehyde (4-methoxybenzaldehyde, for synthesis), and resorcinol were purchased from Sigma-Aldrich, Merck KGaA (Darmstadt, Germany). Silica gel S (particle size: 32–63 μm, 230–400 mesh ASTM) were purchased from Riedel-de Haën. DEAE-cellulose 23 was purchased from Whatman. Polyisobutylmethacrylate was purchased from Sigma-Aldrich. Deionized water (Milli Q) was prepared with Purelab Flex 2 water purification system (AB Ninolab) and all organic solvents were redistilled prior to use.

Hořejší K., Jin C., Vaňková Z., Jirásko R., Strouhal O., Melichar B., Teneberg S, & Holčapek M. (2023). Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues. The Journal of Biological Chemistry, 299(3), 102923.

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Glycolipid Antibody Binding Assay

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Antibody binding to TLC-adsorbed glycolipids was assayed as previously described28 (link). Gangliosides were separated on TLC plates in running solvent chloroform/ methanol/ aqueous 0.2% CaCl₂ (45:45:10), using a tank designed to obtain highly reproducible chromatograms29 . After air-drying, plates were coated by dipping for 2 min in a 0.5% solution of polyisobutylmethacrylate (Sigma, St. Louis, MO) in n-hexane/ chloroform (9:1). Plates were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline containing 0.05% Tween 20 (PBSt) for 1 h, incubated overnight with BSA-PBSt diluted serum (1/200), and washed thoroughly with PBSt. Binding was detected following 2 h incubation with BSA-PBSt diluted (1/1000) peroxidase-conjugated anti-human IgG (γ-chain) antibodies (Sigma). All incubation steps were performed at 4 °C. After washing, color was developed in a substrate solution containing 2.8 mM 4-chloro-1-naphthol and 0.01% H₂O₂ in methanol-20 mM Tris-HCl buffer, pH 7.4 (1:30). The reaction was stopped after 20 min by washing the plates with PBSt. For quantitative studies, spots were measured by densitometry scanning at 590 λ.

Lardone R.D., Yuki N., Irazoqui F.J, & Nores G.A. (2016). Individual Restriction Of Fine Specificity Variability In Anti-GM1 IgG Antibodies Associated With Guillain-Barré Syndrome. Scientific Reports, 6, 19901.

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Glycosphingolipid Fractionation and Antibody Assay

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The isolation and fractionation of glycosphingolipids and the orcinol staining were performed as described previously [22 (link)]. Cellular lipids were extracted with chloroform/methanol from 10⁷ 2102Ep cells. The neutral glycosphingolipids were separated from the phospholipids and gangliosides, purified in peracetylated form, de-O-acetylated, and desalted. Glycosphingolipid samples were solubilized in chloroform/methanol (2:1, v/v), applied to HPTLC plates (Kieselgel 60, Merck, Darmstadt, Germany), and developed with chloroform/methanol/water (55:45:9, v/v/v). The dried plates were immersed in 0.05 % polyisobutylmethacrylate (Aldrich, Steinheim, Germany) in hexane for 1 min, dried, sprayed with TBS (0.05 M Tris buffer, 0.15 M NaCl (pH 7.4)), and blocked in 5 % BSA. For antibody assays, the plates were successively overlaid with 1) primary antibody diluted in TBS/1 % BSA (TBS-BSA) for 1–1.5 h; 2) biotinylated goat anti-mouse Ig antibody (Dako, Glostrup, Denmark), diluted 1:5000 with TBS-BSA; 3) ExtrAvidin-alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:1000 with TBS/ BSA/0.2 % Tween20 for 1 h; and 4) the substrate solution (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, Sigma-Aldrich). Other details were as described previously [22 (link), 26 (link)]. Each HPTLC experiment was repeated three times.

Kaczmarek R., Mikolajewicz K., Szymczak K., Duk M., Majorczyk E., Krop-Watorek A., Buczkowska A, & Czerwinski M. (2016). Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase. Glycoconjugate Journal, 33(6), 963-973.

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Immunostaining of Gangliosides GM1 and GD1a

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To confirm the presence of GM1 and GD1a (respectively mono and disialo- species of gangliosides) on lipid extracts of J774.16 Mφs, we performed an immunostaining assay using monoclonal antibodies to GM1 and GD1a (Schnaar et al., 2002 (link)). GSL were resolved by TLC as above and the plate was dipped in a solution of 0.2% polyisobutyl methacrylate (Aldrich) in hexane for 30s. The plate was dried, sprayed with PBS and blocked for nonspecific binding with 10 mg/ml BSA in PBS for 1 h at room temperature. After washing with PBS three times, the plate was incubated with antibodies to the gangliosides (2 μg/ml) for 2 h at room temperature followed by incubation with a secondary alkaline phosphatase-conjugated anti-mouse IgG (1 μg/ml) under the same conditions. The plate was washed three times with PBS and then developed by using Sigma Fast NBT-BCIP according to the manufacturer’s instructions. Reactive bands (purple) were compared with standards resolved at the same time and conditions, but developed with resorcinol/HCl reagent.

Guimarães A.J., de Cerqueira M.D., Zamith-Miranda D., Lopez P.H., Rodrigues M.L., Pontes B., Viana N.B., DeLeon-Rodriguez C.M., Pereira Rossi D.C., Casadevall A., Gomes A.M., Martinez L.R., Schnaar R.L., Nosanchuk J.D, & Nimrichter L. (2018). Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalization by macrophages.. Cellular microbiology, 21(3), e12976.

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