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High ph fractionation kit

Manufactured by Thermo Fisher Scientific
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The High pH Fractionation Kit is a laboratory tool designed for the separation and purification of biomolecules. It utilizes high pH conditions to fractionate samples and isolate specific components based on their charge properties. The kit provides the necessary reagents and protocols to perform this fractionation process.

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Lab products found in correlation

Microspin columns, by Nest Group (3 mentions) High select fe nta phosphopeptide enrichment kit, by Thermo Fisher Scientific (2 mentions) High select tio2 phosphopeptide enrichment kit, by Thermo Fisher Scientific (2 mentions) Fe nta, by Thermo Fisher Scientific (1 mentions) Ptmscan, by Cell Signaling Technology (1 mentions) Ptmscan phospho enrichment imac fe nta magnetic beads, by Cell Signaling Technology (1 mentions) Tmtpro 16plex, by Thermo Fisher Scientific (1 mentions) Easypep mini ms sample prep kit, by Thermo Fisher Scientific (1 mentions) Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)

5 protocols using high ph fractionation kit

1

Phosphoproteome Analysis Using TiO2 and Fe-NTA

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Before phosphopeptide enrichment, 25 µg of peptide of each sample was set aside for the proteome measurement. A High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Scientific, Waltham, MA, USA) was used to enrich phoshphopeptides followed by a second enrichment step for the flowthrough fraction of the TiO2 kit using the High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Afterwards, enriched phosphopeptides were desalted by C18 spin tips. BCA assay was used before labeling to estimate the peptide concentration of each sample. Equal amounts of enriched phosphopeptides and peptides for the proteomic analysis were labeled and combined as previously described [52 (link)]. After labeling, peptides were fractionated into eight fractions utilizing a high-pH fractionation kit (Thermo Scientific, Waltham, MA, USA). For the analysis of the effect of the pan-RAS inhibitor on the phosphoproteome, no fractionation was performed. Finally, peptides were vacuum dried and stored at −80 °C until LC–MS measurement.
Stieglitz F., Gerhard R., Hönig R., Giehl K, & Pich A. (2022). TcdB of Clostridioides difficile Mediates RAS-Dependent Necrosis in Epithelial Cells. International Journal of Molecular Sciences, 23(8), 4258.
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2

Cell Lysis and Phosphopeptide Enrichment

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2 × 108 cells per time point were harvested by centrifugation at 1,000 × g for 10 min at 4 0C, and the supernatant removed. Following a wash with 1 × PBS, cells were lysed at 1 × 109 cells/ml in ice-cold lysis buffer (0.1 μM TLCK, 1 μg/ml Leupeptin, Phosphatase Inhibitor Cocktail II (Calbiochem), 1 mM PMSF, 1 mM Benzamidine) for 5 min at RT before transfer to -80°C for storage. Briefly, samples were combined according to the experimental regimen, subjected to FASP and tryptic digest [18 (link), 22 (link)], and peptides separated from phosphopeptides using Fe-IMAC was performed as described by Ruprecht et al [23 (link)]. Samples were fractionated using the High pH Fractionation kit (ThermoFisher), and the eluates concatenated into five fractions. Further details of sample processing can be found in S1 Methods.
Benz C, & Urbaniak M.D. (2019). Organising the cell cycle in the absence of transcriptional control: Dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally. PLoS Pathogens, 15(12), e1008129.
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3

Comprehensive Proteomics Analysis of Trigeminal Ganglion

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Trigeminal ganglion tissue from both Tgp35 and wildtype mice homogenates were lysed in 5% SDS in 10 mM sodium phosphate buffer, and then boiled for 2 minutes. Samples were clarified by centrifugation at 14,000g for 10min, and the supernatants were collected. Total protein concentration was measured using a BCA Protein Assay Kit. After reduction with TCEP and alkylation with iodoacetamide, proteins were digested with trypsin following the S-Trap digestion protocol (HaileMariam et al., 2018 (link)). The resulting tryptic peptides were dried under vacuum, labeled with TMTPro (A44520) following the manufacturer’s protocol. 10% of each sample was set aside for analysis of background proteome and the remaining combined for phosphopeptide enrichment.
For phosphopeptide enrichment, we incorporated a 3-step sequential enrichment process: first with PTMScan from Cell Signaling Technology, followed by TiO2 enrichment, then Fe-NTA from Thermo Scientific. All steps followed the manufacturer’s recommendations. 1 μg of enriched peptide from each step was analyzed, and after that the 3 fractions were combined and fractionated with the high pH fractionation kit from Thermo Scientific. Background proteome samples were combined and fractionated with the same protocol.
Hu M., Doyle A.D., Yamada K.M, & Kulkarni A.B. (2022). Visualization of trigeminal ganglion sensory neuronal signaling regulated by Cdk5. Cell reports, 38(10), 110458.
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4

Affinity-Purified Protein Digestion

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Affinity-purified samples in Urea (8M) were reduced with 5 mM TCEP (30 min at 37 °C) and alkylated with (10 mM IAA at room temperature). Urea was diluted to a concentration of 5.5 M for Lys-C proteolysis (Wako) (0.4 µg, 3 h) and trypsin proteolysis (Promega) (1.2 µg, overnight). Digested samples were acidified by addition of formic acid, cleaned up with microspin columns (The Nest Group), and subjected to high pH fractionation, with a procedure based on the high pH fractionation kit by Thermo.
Ciuffa R., Uliana F., Mannion J., Mehnert M., Tenev T., Marulli C., Satanowski A., Keller L.M., Rodilla Ramírez P.N., Ori A., Gstaiger M., Meier P, & Aebersold R. (2022). Novel biochemical, structural, and systems insights into inflammatory signaling revealed by contextual interaction proteomics. Proceedings of the National Academy of Sciences of the United States of America, 119(40), e2117175119.
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5

Comprehensive Proteome and Phosphoproteome Analysis

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Tissues were prepared for proteomic analysis using EasyPep Mini MS Sample Prep Kit according to manufacturer’s instructions (Thermo Fisher Scientific). Protein concentration was measured using Pierce BCA Protein Assay (Thermo Fisher Scientific). 100 μg aliquots of protein from each sample were reduced, alkylated, and digested. Remaining protein extracts were saved for electrophoresis and western blot. Samples were labeled with TMTpro 16-plex (Thermo Fisher Scientific) according to the manufacturer’s instructions. 10 μg of each sample was combined, fractionated using high pH fractionation kit (Thermo Fisher Scientific), and used for full proteome analysis. The remaining 90 μg of each sample was pooled for phospho-peptide enrichment with PTMScan Phospho-Enrichment IMAC FE-NTA Magnetic Beads (Cell Signaling Technology) according to the manufacturer’s instructions. The flow through was then enriched according to the High-Select SMOAC protocol (Thermo Scientific Scientific). In the SMOAC protocol, samples were sequentially enriched with High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) and High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific).
Doty M., Yun S., Wang Y., Hu M., Cassidy M., Hall B, & Kulkarni A.B. (2022). Integrative multiomic analyses of dorsal root ganglia in diabetic neuropathic pain using proteomics, phospho-proteomics, and metabolomics. Scientific Reports, 12, 17012.
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