Guanidine hcl

Manufactured by Thermo Fisher Scientific

Sourced in United States

Guanidine HCl is a chemical compound commonly used in molecular biology and biochemistry applications. It serves as a denaturant, disrupting the non-covalent interactions within proteins and nucleic acids, which can facilitate tasks such as protein purification and nucleic acid extraction.

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Close spelling variants of this product

Guanidine HCl (Gu-HCl), 8M Guanidine HCl

8 protocols using guanidine hcl

Enzymatic Synthesis of Erythrulose

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TPP, MgCl₂, glycolaldehyde (GA) and Erythrulose [Ery] were purchased from Sigma-Aldrich; Tris-HCl was purchased from VWR International and Guanidine-HCL was purchased from Life Technologies Ltd. HPA was synthesised from bromopyruvic acid and LiOH, as described previously^{31 (link)}.

Wilkinson H.C, & Dalby P.A. (2020). The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation. Scientific Reports, 10, 4148.

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Enzymatic Synthesis of Erythrulose

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TPP, MgCl₂, glycolaldehyde (GA) and erythrulose [Ery] were purchased from Sigma-Aldrich; Tris-HCl was purchased from VWR International and Guanidine-HCL was purchased from Life Technologies Ltd. HPA was synthesised by reacting bromopyruvic acid with LiOH, as described previously^{41 (link)}.

Wilkinson H.C, & Dalby P.A. (2019). Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations. Scientific Reports, 9, 16116.

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Quantitative ATP Measurement Assay

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ATP levels were measured using the ATP determination kit (A22066; Thermo Fisher Scientific). Five adult males of a specified age were homogenized in 100 μl of 6 M guanidine HCl (24115; Thermo Fisher Scientific) and 0.01 M Tris-HCl (pH 7.3) and frozen in liquid nitrogen for 5 min. The sample was then incubated at 95°C for 5 min and centrifuged at 12,000 × g for 10 min at 4°C. 10 μl of the sample was used for each 100 μl reaction as instructed in the kit manual. ATP levels for each sample were normalized to protein concentration measured using the Pierce BCA protein assay kit.

Klucnika A., Mu P., Jezek J., McCormack M., Di Y., Bradshaw C.R, & Ma H. (2022). REC drives recombination to repair double-strand breaks in animal mtDNA. The Journal of Cell Biology, 222(1), e202201137.

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Testicular ATP Quantification

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ATP levels were measured using the ATP determination kit (ThermoFisher Scientific, A22066). For each genotype, 10 testes were dissected from 1 to 2-d-old males and homogenized in 100 μL of 6 M guanidine HCl (ThermoFisher Scientific, 24115) and 0.01 M Tris-HCl (pH 7.3), and frozen in liquid nitrogen for 5 min. The sample was then incubated at 95 °C for 5 min and centrifuged at 12,000 × g for 10 min at 4 °C. 10 μL of the sample was used for each 100 μL reaction as instructed in the kit manual. ATP levels for each sample were normalized to protein concentration measured using the Pierce BCA protein assay kit (ThermoFisher Scientific, 23225). For each genotype, six replicates were performed.

Li A.Y., Di Y., Rathore S., Chiang A.C., Jezek J, & Ma H. (2023). Milton assembles large mitochondrial clusters, mitoballs, to sustain spermatogenesis. Proceedings of the National Academy of Sciences of the United States of America, 120(34), e2306073120.

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Glycoprotein Extraction and N-Glycan Analysis

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10⁷ cells per each cell line were collected. Allantoic and amniotic membranes were extracted from 10-day old chicken embryos. All samples were treated as described previously^{37 (link),59 (link)}. Briefly, each cell line was subjected to sonication in the presence of detergent 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, #10810118001, Roche), reduction in 4 M guanidine-HCl (#24115, Thermo), carboxymethylation, and trypsin (#T0303, Sigma) digestion. The digested glycoproteins were then purified by plus short HLB-Sep-Pak (#186000132, Waters Corp.). N-glycans were released by peptide N-glycosidase F (E.C. 3.5.1.52; #11365177001, Roche) digestion. Released N-glycans were permethylated using the sodium hydroxide procedure and purified by classic short C₁₈-Sep-Pak (#WAT051910, Waters). Permethylated N-glycans were eluted at the 50% acetonitrile fraction.

Kikuchi C., Antonopoulos A., Wang S., Maemura T., Karamanska R., Lee C., Thompson A.J., Dell A., Kawaoka Y., Haslam S.M, & Paulson J.C. (2023). Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines. Nature Communications, 14, 6178.

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Biotinylated Protein Enrichment and Purification

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Prior to incubation with protein sample, 100 μL of settled NeutrAvidin agarose resin (Pierce Biotechnology, Rockford, IL) was blocked by incubation with 2.5% biotin-free BSA (Sigma, St. Louis, MO) in 0.1 M PBS for 1 h at room temperature. The blocking solution was removed and the protein samples (200 μL) were incubated with the NeutrAvidin agarose resin for 2 h at room temperature. The resin was washed 5 × 20 min in 0.1 M PBS. After washing all of the unbound protein, the biotinylated proteins were removed from the resin by 4 × 20 min incubations in 150 μL of the elution buffer: 8 M guanidine-HCL (ThermoFisher Scientific), pH 1.5. All of the elution buffer fractions were collected and combined to ensure maximum protein recovery from the resin. The entire 500 μL volume of elution buffer containing the biotinylated proteins was immediately dialyzed against 0.1 M PBS for 24 h at 4°C. The eluates were precipitated from the PBS via trichloroacetic acid (TCA) protein precipitation using deoxycholate. The precipitated protein pellets were resuspended in 50 μL 0.1 M PBS and stored at −20°C. Protein concentration was determined by BCA analysis (Thermo Scientific, Rockford, IL).

Tooker R.E, & Vigh J. (2015). Light-evoked S-nitrosylation in the retina. The Journal of comparative neurology, 523(14), 2082-2110.

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Heparin and Heparan Sulfate Binding Assays

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Porcine-extracted heparin sodium salt (MW_avg = 15 kDa, polydispersity = 1.4) and heparan sulfate (MW_avg = 14 kDa) was purchased from Celsus Laboratories (Cincinnati, OH, United States). Low molecular weight heparin (MW_avg = 4.5 kDa) was purchased from Iduron (Manchester, United Kingdom). Heparin oligosaccharides and de-sulfated heparins (deNS, de2S, de6S) were purchased from Iduron (Alderly Park, Edge, Chesire, United Kingdom). Chemically defined oligosaccharides were prepared using a chemobiocatalytic approach with heparosan starting material as described in Yu et al. (Yu et al., 2022 (link)). EZ-Link NHS-PEG-4 purchased from Thermo Fisher Scientific (Waltham, MA, United States). Streptavidin (SA) sensor chips and HBS-EP buffer were purchased from Cytiva (Marlborough, MA, United States). SPR measurements were performed on a Biacore 3,000 (Cytiva, Marlborough, MA, United States). Tris-HCl, guanidine HCl, urea, HEPES disodium salt, EDTA, NaCl, surfactant P20/Tween 20, glycine, potassium thiocyanate, and magnesium chloride were purchased from Thermo Fisher Scientific, ACROS Organics (Pittsburgh, PA, United States), and Sigma Aldrich (Burlington, MA, United States). The HIS Lite™ OG488-Tris NTA-Ni complex was purchased from AAT Bioquest (Sunnyvale, CA, United States).

Gandy L.A., Canning A.J., Lou H., Xia K., He P., Su G., Cairns T., Liu J., Zhang F., Linhardt R.J., Cohen G, & Wang C. (2022). Molecular determinants of the interaction between HSV-1 glycoprotein D and heparan sulfate. Frontiers in Molecular Biosciences, 9, 1043713.

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Glycan Release and Purification Protocol

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Trypsin/Lys‐C mixture was obtained using a Mass Spec Grade (code V5071, Promega Corporation, Madison, WI, USA). Tris–HCL (100 mM, pH 7.0) buffer was prepared by TRIS Merck (code 1.08382.1000) at pH 7.0 with diluted HCl prepared from HCl 37% obtained from VWR BDH (code 20255.290, Avantor VWR International Ltd. BDH Chemicals); 8 M guanidine HCl was purchased from ThermoFisher Scientific (code 24115). Purified water was obtained by a Milli Q System Millipore mod. Advantage A10; 500 mM maleimide was prepared using maleimide obtained from Sigma (code I29585). N‐Glycanase was purchased from Agilent Technologies (formerly Prozyme, Santa Clara, CA, USA), code GKE‐5006D. Ultrafiltration device Amicon Ultra‐4 10K was obtained from Merck Millipore (code UFC801096). Amicon Ultra‐4 10K was pre‐washed with 1000 μL of MQ water and a final volume of 100 μL of MQ water. Hydrolyses were performed at 37°C on an Eppendorf thermomixer mode comfort (Eppendorf, a division of Merck KGaA). Formic acid for ultra‐high‐performance liquid chromatography (UPLC) mobile phases was purchased from Merck (code 1.00264.1).

Datola A., Melchiorre M., Baroni F., Iozzino L, & Palmese A. (2022). Characterization of disulfide bridges pattern of recombinant human interleukin 12 fusion protein p40 subunit and identification and quantification of cysteinylated free cysteine 252. Rapid Communications in Mass Spectrometry, 36(14), e9313.

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