Hi,
1. I've isolated the pure mitochondria and ER vesicles through ultracentrifugation.
2. I can observe them clearly under microscope in the liquid fractions.
3. but I want to fix them and stain with organelle specific antibody.
4. I used Poly-D-Lysine coated coverslips to spread the fractions. but the fractions are lost during washing steps.
Can any one suggest me the best way to fix and stain the cellular fractions?
Hi Santosh
Poly-D-Lysine coated slips are good for attachment of cell membrane with negative charges
In case of cell fractions, you can try other alternatives like ECM, matrigel, collagen or laminin to coat the coverslips. These can be the best way to spread the fraction on coverslips.
Also, fixing with methanol can help.
Perhaps easiest is to spot-blot the fractions (1-2 µL) onto a PVDF-membrane and then develop just like a western blot.
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