CD138+ cells were lysed with M-Per buffer (Thermo Fisher Scientific, Rockford, IL) containing phosphatase and protease inhibitors. Approximately 50–72 ng of protein was used per sample, and experiments were performed using a Nanopro1000 instrument (ProteinSimple, Santa Clara, CA), as previously described (29 (link), 30 (link)). Various phosphorylated isoforms of MEK and ERK were detected using anti-phospho-ERK (Cell Signaling Technology, Danvers, MA), anti-MEKpS218/222 (Epitomics, Burlingame, CA), anti-MEKpT292 (Millipore), anti-MEKpT386 (Novus Biologicals, Littleton, CO), anti-MEKpT394 (Abcam, Cambridge, MA), anti-MEKpS298 (Cell Signaling Technology), and anti-Beta2-microglobulin (Abcam) primary antibodies with HRP-conjugated goat anti-rabbit or goat anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies. Luminol and peroxide (ProteinSimple) were added to generate chemiluminescent light. The digital images were analyzed and quantified with Compass software (ProteinSimple). ERK and MEK were normalized to Beta2-microglobulin loading controls.
Anti beta2 microglobulin
Manufactured by Abcam
Sourced in United States
Anti-Beta2-microglobulin is a laboratory product used to detect and measure the levels of beta-2-microglobulin, a protein found on the surface of many cells. It is a key component in the major histocompatibility complex (MHC) class I molecule.
Automatically generated - may contain errors
Lab products found in correlation
M per buffer, by Thermo Fisher Scientific (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit or goat anti mouse, by Jackson ImmunoResearch (1 mentions)
Anti yes, by BD (1 mentions)
Anti erp57, by Abcam (1 mentions)
Anti beta actin, by MP Biomedicals (1 mentions)
Anti caveolin 1, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Anti flotillin 1, by Merck Group (1 mentions)
Anti calreticulin, by Abcam (1 mentions)
Hrp conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti beta2 microglobulin
1
Quantifying Phosphorylated MEK and ERK
2
Profiling Antigen Presentation Machinery
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The protein concentration of each fraction was measured by a Bradford protein assay. An amount of 5 µg protein of each fraction was separated on 10% polyacrylamide gels at 40 mA per gel for 2 h and were blotted onto nitrocellulose membranes by semi-dry Western blot at 180 mA per membrane for 1 h. Membranes were blocked in 5% skim milk powder in TBS-T, incubated with the indicated primary antibody solution (in TBS-T supplemented with 5% BSA and 0.1% sodium azide) at 4 °C overnight followed by incubation with corresponding HRP-conjugated secondary antibody solution (in 3% BSA in TBS-T) at RT for 1 h. Proteins were visualized with ECL Detection Reagents (GE Healthcare, Chicago, IL, USA). The antibodies used were anti-beta Actin (MP, Irvine, CA, USA), anti-Tapasin, anti-beta2-Microglobulin, anti-ERp57, anti-Calreticulin (all Abcam, Cambridge, UK), anti-Yes (BD Bioscience), anti-Flotillin1, anti-Caveolin1 (Sigma-Aldrich, Seelze, Germany), anti-TAP1 (Novus Biologicals, Cambridge, UK), anti-TAP2 (MBL, Woburn, MA, USA), HRP-conjugated goat anti mouse and HRP-conjugated goat anti-rabbit (Thermo Scientific, Bonn, Germany).
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