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BODIPY-FL-ceramide from invitrogen for Golgi staining - Question on staining protocol (Sep/06/2006 )

Hi

I just got the golgi probe from invitrogen and wondered if anyone has used it before. The product protocol says to incubate cells at 4oC with the probe in HBSS/HEPES for 30min and at 37oC in media for another 30min.

I was wondering:
1) can i use PBS instead of HBSS/HEPES
2) what might the 4oC incubation be for? can i just incubate it straight at 37oC?

Thanks!

-xtrios-

QUOTE (xtrios @ Sep 7 2006, 01:30 AM)
Hi

I just got the golgi probe from invitrogen and wondered if anyone has used it before. The product protocol says to incubate cells at 4oC with the probe in HBSS/HEPES for 30min and at 37oC in media for another 30min.

I was wondering:
1) can i use PBS instead of HBSS/HEPES
2) what might the 4oC incubation be for? can i just incubate it straight at 37oC?

Thanks!


I've used it before but I just used HBSS. And come to think of it I have no idea what the 4oC incubation is for. Perhaps it helps permeabilize the cells for uptake of the ceramide but really I have no idea. I would suggest calling Invitrogen and asking them. They should know or at least be more helpful than me. laugh.gif

-lab_geek-

Hi,

About the 4°C incubation:
Ceramide at 4°C is inserted into the Plasma Membrane, as temperature is increased lipid transport is active transporting the Ceramide via small vesicles to the ER. From there CERT is transporting the Ceramide to the Golgi. So make sure that you wait long enought until you get distinct Golgi staining. After that Ceramide is transported to the Plasa Membrane again.

Hope this could be helpful.

For further reference check CERT papers from Hanada and
PHOSPHATIDYLINOSITOL 4-KINASE IIIβ REGULATES THE TRANSPORT
OF CERAMIDE BETWEEN THE ENDOPLASMIC RETICULUM AND GOLGI from Balla, Toth (JBC 2006)

-Azrael-