To conduct proteomics of lysosomes, I am trying to replicate the "Lyso-IP" protocol described in the paper ; G.A. Wyantet al., Science10.1126/science.aar2663 (2018).
Unfortunately, I can not isolate "pure" lysosomes. My Western Blot images show contamination of ER proteins (calreticulin and calnexin) and few of mitochondria protein (VDAC) though negative controls (cells not expressing TMEM 192-HA) does not show such bands. I can see enrichment of TMEM192 after IP.
To reduce the contamination, are there any tips?
I use the HA-tagged beads (Thermo) and the same buffer indicated in the paper. I do not use homogenizer, rather I extract cells using 27G syringe. Thank you!
Gentle homogenization could reduce damage to organelles, thus, contamination in immunoprecipitation. Question I have is- are you using the same cells used in the paper? if not, you may need optimization.
Thanks Divaker. Actually, I use different cells (immune cells). I understand I should optimize conditions(buffer or homogenization?)
Foozhan Tahmasebinia
Thank you for useful information! If you don't mind, could you share such protocol?(any papers?)
Kohei Tsujimoto Hi Kohei, did the CaCl2 step help? I am also using a similar method for Lyso-IP using the Thermo HA-tag magnetic beads, but I do not understand why do I have mitochondrial contamination ( I get less of ER contamination, but lot of mito contamination). I used a LAMP1-HA construct for the lysosomes and homogenized the cells.
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