To immunopurified lysosomes, the Lyso-IP method was used with some modifications50 (link). HEK293T cells stably expressing TMEM192-FLAG (control) or TMEM192-HA were collected and resuspended in ice-cold KPBS (136 mM KCl, 10 mM KH2PO4, adjusted to pH 7.25 with KOH) buffer and homogenized using dounce homogenizer (~20 strokes). The homogenized cells were gently collected and centrifuged for 2 min at 1000×g. The supernatant obtained was incubated with anti-HA antibodies-conjugated-agarose beads (Sigma-Aldrich) at 4 °C for 15 min. Beads were gently washed thrice with KPBS, and bound lysosomes were eluted in Laemmli buffer and subjected to SDS-PAGE for further analysis.
Anti ha antibodies conjugated agarose beads
Manufactured by Merck Group
Anti-HA antibodies-conjugated-agarose beads are a laboratory product that consists of agarose beads covalently linked to anti-HA (hemagglutinin) antibodies. The core function of this product is to facilitate the purification and detection of HA-tagged proteins in various biochemical and cell-based experiments.
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Lab products found in correlation
2 protocols using anti ha antibodies conjugated agarose beads
1
Purification of Lysosome-Enriched Fraction
2
TMEM192-HA Lysosome Immunopurification
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To immunopurify lysosomes, the "LysoIP" method (Abu-Remaileh et al., 2017) was used with some modi cations. HEK293T cells stably expressing TMEM192-FLAG (control) or TMEM192-HA were collected and resuspended in ice-cold KPBS (136 mM KCl, 10 mM KH 2 PO 4 , adjusted to pH 7.25 with KOH) buffer and homogenized using dounce homogenizer (~ 20 strokes). The homogenized cells were gently collected and centrifuged for 2 min at 1000xg and the supernatant obtained after centrifugation was incubated with anti-HA antibodies-conjugated-agarose beads (Sigma-Aldrich) at 4°C for 15 min. Beads were gently washed thrice with KPBS, and bound lysosomes were eluted in Laemmli buffer and subjected to SDS-PAGE for further analysis.
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