MDA-MB-231 cells were lysed in buffer [20 mM Tris–HCl, pH 7.3, 300 mM KCl, 0.2 mM EDTA, 0.5% Triton, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM glycerophosphate, and 0.1 mM Na3VO4, and protease inhibitors]. Total protein (50 mg) was IP with anti-ITCH (1:1000; BD Biosciences) or unspecific mouse monoclonal IgG antibodies (I-1000; GeneTex Inc.) and 50 μl protein G-conjugated beads (Thermo Fisher), as above. Washed beads were separated by SDS-PAGE. Following Coomassie Blue Staining, protein bands were excised and digested. After separation on a reversed phase LC column, eluted peptides were analyzed on a MALDI-QIT-TOF-based mass spectrometer with electrospray ionization (Micromass/Waters). The MS/MS data were processed using Masslynx software (Micromass), and the MASCOT (Matrix Science) search engine was used to search the NCBI non-redundant database. Protein identifications were based on a minimum random probability score of 25 and with a mass accuracy of 0.1 Da.
Anti itch
Manufactured by BD
Anti-ITCH is a laboratory equipment designed to measure and analyze the itch response in various experimental settings. It provides accurate and reliable data on itch-related parameters, enabling researchers to study the underlying mechanisms and potential treatments for conditions associated with itching.
Automatically generated - may contain errors
Lab products found in correlation
Protein g conjugated beads, by Thermo Fisher Scientific (1 mentions)
Unspecific mouse monoclonal igg antibodies 1 1000, by GeneTex (1 mentions)
Anti tubulin, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti c myc, by Merck Group (1 mentions)
Gateway cloning technology, by Thermo Fisher Scientific (1 mentions)
Mg132, by R&D Systems (1 mentions)
Pet dest42, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Anti gapdh monoclonal antibody, by Abcam (1 mentions)
Anti txnip, by Abcam (1 mentions)
Anti ubiquitin, by Abcam (1 mentions)
Anti vlcad, by Abcam (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab19247, by Abcam (1 mentions)
4 protocols using anti itch
1
Mass Spectrometry Identification of ITCH Interactome
2
Proteasome Inhibition and Protein Analysis
3
Quantifying Protein Expression in Fibroblasts
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Western blotting of whole cell lysates from patient and control fibroblasts was performed as previously described [20 ]. Twenty five micrograms of protein were loaded onto a 4–15% gradient precast SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA). Following electrophoresis separated proteins were transferred onto a nitrocellulose membrane. Primary antibodies included anti-ITCH (1;1000, BD Transduction Laboratories, San Jose, CA, #611198), Anti-VLCAD (1:1000, antigen produced by the Vockley Lab, UPMC Children's Hospital of Pittsburgh and antibody generated by Cocalico Biologics, Stevens, PA), Anti-TFP α/β (1:2000, produced by the Vockley Lab), anti-Ubiquitin (1:1000, Abcam, Cambridge, MA, #ab19247), and anti-TXNIP (1:500, Abcam, #ab188865). Blots were incubated with HRP conjugated secondary antibodies. Primary purified mouse anti-GAPDH monoclonal antibody (1,25,000, Abcam, #ab8245) was used as a loading control (Supplemental Table S1). All western blot studies were performed more than once from different celluar lysates (technical replicates).
4
Immunofluorescence Imaging of ITCH Localization
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Patient and control fibroblasts were seeded in triplicates on tissue culture-treated glass coverslips at a density of 25,000 cells per coverslip and allowed to grow overnight at 37 °C in 5% CO2, 95% humidity. Cells were fixed, permeabilized, and blocked as previously described [24 (link)]. For ITCH co-localization studies, primary antibodies used include anti-ITCH (1:50, BD Transduction Laboratories, San Jose, CA, # 611198), and anti-ubiquitin (1:50, abcam ab19247). Primary antibody incubation was followed by incubation in an appropriate Alexa Fluor secondary antibody (Invitrogen, Waltham, MA,1:1000) for 1 h at room temperature. DAPI was used for visualization of nuclei. Coverslips were then mounted and imaged on a Zeiss LSM 710 Confocal microscope (ZEISS, White Plains, NY) at 63× magnification.
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