Hello, I am currently using antibody-coated magnetic beads to isolate mitochondria and then peroxisomes from the same sample. But I can't get rid of peroxysomes in the mitochondrial fraction.
Apparently ETDA, EGTA or salts in the homogenehization buffer can not help.
But would a little bit of mild detergent be efficient ?
Dear Sarah,
The answer to your question can be found in this paragraph taken from a paper entitled "Proteomic Analysis of Rat Liver Peroxisome PRESENCE OF PEROXISOME-SPECIFIC ISOZYME OF LON PROTEASE* " in First Published on October 15, 2003, doi: 10.1074/jbc.M305623200
January 2, 2004 The Journal of Biological Chemistry, 279, 421-428.
mmunopurification of Peroxisomes—Anti-PMP70 specific antibody was bound to magnetic beads (1×108), using an anti-rabbit Fc fragment antibody as a linker antibody according to the manufacturer's instruction. The peroxisome-enriched fractions from the Nycodenz density gradient (about 500 μg of protein) were incubated with the magnetic beads suspended in phosphate-buffered saline containing 2 mM EDTA at 4 °C for 6 h. After incubation, bead complexes were collected by placing the reaction tube in a magnet stand. The complex was washed three times with phosphate-buffered saline containing 2 mM EDTA. Peroxisomes were eluted by boiling in 200 μl of 2× SDS sample buffer for SDS-PAGE (80 mM Tris, 4% SDS, 10% sucrose, 0.002% bromphenol blue, and 20 mM dithiothreitol, pH 6.8). The supernatant separated by placing in a magnet stand was subjected to SDS-PAGE. Peroxisomal membranes were obtained by incubating the bead-bound peroxisomes with 0.1 M Na2CO3 for 30 min at 4 °C. Bead complexes were washed twice with 0.25 M sucrose, 2 mM EDTA, and 0.1% ethanol, and eluted with the SDS sample buffer as above.
Hoping this will be helpful,
Rafik
what are the source and the amount of starting material? what do you want to do with a prep?
however, depending on your source you can try various types of gradient centrifugation.
Dear Sara, evidently you use liver for the isolation of mitochondria. In principle, but with liver in particular, because brain or heart mitochondria are not contaminated much with peroxisomes, the easiest way to get rid of peroxisomal and/pr lysosomal fraction is to cut off light fractions by sedimenting mitochondria at maximum 8000 g. The first centrifugation (to remove cell debris and erythrocytes) perform at 9500-100 g. This will also remove some heavy fraction of mitochondria.
The basics are described in my book "Practical mitochondria? on my page in ResearchGate.
Sincerely,
Alexander Panov
I am using cell culture material. A 175 msquare flasque.
A precedent colleague did not manage to achieve good purity of peroxisomes via centrifugation in a percolle gradient so I am now trying a new technique (magnetic beads).
the fractions (nuclear, mitochondrial and peroxisomal) will be analyzed by western blotting.
thanks for your help !
In your cell cultures, do you use streptomycin?
Regarding the cell's mitochondria, namely their functional state. Please, check on google or on Quertle search engine two words: "mitochondria and streptomycin".
Personally, I do not believe any works on cells performed in the presence of stre[streptomycin.
Good luck.
Hello Sara! I have the same problem you did back then! Could you find a solution ?
Hello Magali,
no, an other part of my research was working much better so we put all our forces in that part instead of finalizing this magnetic beads protocol.
Good luck then !! Could you tell me if you succeed !! =)
thanks in advance !