The recombinant protein band was manually excised from the Coomassie blue-stained gel and destained by incubating with 200 μL of destaining solution for 30 min at 37°C with shaking. The gel slice was digested and extracted using the Agilent Protein In-gel Trypsin Digestion Kit (Agilent, USA). The sample was then cleaned up using ZipTipC18 pipette tips (Zip-Tip U-C18; Millipore, Bedford, MA, USA), eluted, and concentrated using a vacuum concentrator (Eppendorf, Westbury, NY, USA) until completely dry. The protein was then analyzed using MALDI-TOF-TOF 4800 (ABSCIEX, Foster City, CA, USA).
Ziptip u c18
Manufactured by Merck Group
Sourced in United States
The ZipTip U-C18 is a laboratory equipment product from Merck Group. It functions as a pipette tip for sample preparation and purification in analytical procedures. The ZipTip U-C18 features a reversed-phase sorbent designed for the extraction and concentration of analytes from complex sample matrices.
Automatically generated - may contain errors
Lab products found in correlation
4800 maldi tof tof, by AB Sciex (1 mentions)
Vacuum concentrator, by Eppendorf (1 mentions)
Sepharose 4b, by GE Healthcare (1 mentions)
Sep pak tc18 cartridge, by Waters Corporation (1 mentions)
Octyl β d glucopyranoside, by Dojindo Laboratories (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
3 protocols using ziptip u c18
1
Protein Isolation and Identification
2
Peptide-HLA Class I Isolation and Analysis
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An established protocol was followed with slight modifications.26 (link),56 (link) A frozen cell pellet of 1.0 × 109 HCT15/β2 m cells was lysed with buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, protease inhibitor cocktail (Sigma), 1 mM PMSF, and 1% octyl-β-D glucopyranoside (Dojindo). The peptide-HLA class I complexes were captured by affinity chromatography using a purified pan-HLA class I monoclonal antibody (W6/32, ATCC) coupled to CNBr-activated Sepharose 4B (GE Healthcare). The peptides bound to HLA class I were eluted with mild acid (0.2% TFA) and desalted using a Sep-Pak tC18 cartridge (Waters) with 28% ACN in 0.1% TFA and ZipTip U-C18 (Millipore) with 50% ACN in 1% FA. Samples were dried using vacuum centrifugation and resuspended in 5% ACN in 0.1% TFA for LC-MS/MS analysis.
3
Protein Sample Preparation Protocol
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