Cells were fixed in 100% methanol on ice for 10 min, washed four times with PBS, and blocked in PBS containing 3% BSA, 0.1% Triton X‐100 (PBS‐TB) for 1 h at room temperature. Cells were incubated for 2 h with the indicated antibodies as the first antibodies, and for 1 h with Alexa Fluor 488‐conjugated anti‐rabbit or mouse IgG or Alexa Fluor 568‐conjugated anti‐rabbit or mouse IgG (Life Technologies) as the second antibodies with Hoechst 33342 (Life Technologies). Fluorescent images were obtained using a BZ‐9000 (Keyence, Osaka, Japan). The antibodies used were: multi‐ubiquitin (MBL, D058‐3), ubiquitin, Lys48‐Specific (Millipore; 05‐1307), TACC3 (Cell Signaling, 8069), LAMP2 (BD Biosciences, 555803), PMP70 (Abcam, Cambridge, UK; ab85550), GM130 (Abcam, ab52649), COX IV (Cell Signaling, 4850).
Lys48 specific
Manufactured by Merck Group
Sourced in United Kingdom, Switzerland
Lys48‐Specific is a laboratory equipment product. It is designed to detect and analyze specific protein modifications. The core function of this product is to identify and quantify the Lys48 ubiquitination site on target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Lamp2, by BD (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Tacc3, by Cell Signaling Technology (1 mentions)
Pmp70, by Abcam (1 mentions)
Gm130, by Abcam (1 mentions)
Hrp conjugated anti ha antibody, by Roche (1 mentions)
2 protocols using lys48 specific
1
Immunofluorescence Staining Protocol
2
Ubiquitination of TACC3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HT1080 cells were transfected with pCMV5-FLAG-TACC3 and pcDNA3-HA-ubiquitin for 40 h The cells were then incubated with the indicated compounds in the presence of MG132 (25 μM) for 3 h before being harvested and lysed in SDS lysis buffer. The cell lysates were boiled for 10 min, diluted 10 times with IP lysis buffer (10 mM Hepes at pH7.4, 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA and 1% NP-40) and immunoprecipitated with anti-FLAG agarose-conjugated beads. The precipitates were extensively washed and analyzed by western blotting using an HRP-conjugated anti-HA antibody (Roche, Basel, Switzerland) or anti-ubiquitin, Lys48-Specific (Millipore, 05-1307).
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