Hi everyone! I am trying to perform western blots on extracellular vesicles collected after ultracentrifugation of plasma samples. Briefly, I am diluting 250 ul of platelet-free plasma in 2 ml PBS, UC 2h at 100,000g at 4°C, washing the pellet with 2 ml PBS, and re-ultra centrifugating 2h at 100,000g at 4°C. After that, I am lysing the pellet and running the WB. However, the only thing I can see are aspecific bands recognized by secondary antibodies, corresponding to the light and heavy chains of IgG that are co-precipitated with exosomes. They appear so quickly that I can't look at my bands of interest (eg, CD9, which is about 20 kDa and pretty close to the aspecific band of the light IgG). I do see those aspecific bands whatever species the secondary antibodies are raised against, and also when there are pretended not to recognized the denatured IgG ("Trueblot secondary Ab"). Does anyone have a suggestion on how to prevent such recognition of aspecific bands? NB: I am using TBS-T with 5% non fat dry milk as suggested in the antibody's datasheets...Thank you for your help!
Hi,
Unfortunately, ultracentrifuge -derived micro vesicles and exosomes are almost always heavily contaminated with serum albumin and immunoglobulins unless they are isolated using iodixanol or sucrose gradients, which are not particularly suitable for small volumes of sample. I ll suggest to use ExoQuick precipitation method. There are several publications on PEG mediated precipitation of micro vesicles and exosomes, something you may want to try. PEG precipitation may be a cheaper alternative to ExoQuick reagent that as per my understanding contains PEG or its derivatives.
Hi,
one possible course of action is not to use secondary antibodies for detection of the primary antibodies. One way could be to use biotinylated primary antibodies and do the detection via Streptavidin/Neutravidin-HRP (it is relativly easy to biotinylate IgGs, but in my experience around 10-20% of all antibodies have more or less severe problems to detect their targets after biotinylation) or use one of the commercial reagents which only detect native and not denatured IgGs;like this
https://www.thermofisher.com/order/catalog/product/21232
But be careful, this reagents are not so good in detecting polyclonal rabbit IgGs, they are better with monoclonal mouse IgGs.
Good luck,
Carsten
I would like use your question to ask Syed Ali and Casrten Schmidt one thing:
Do you guys think Anaïs could pre clear the lysate using a protein G resin? In theory it would bind and remove IgG.
Hi Andre,
you could do this, the success depend on how many of the IgGs will bind to the Protein G resin, maybe Protein A/G would be more efficient. I don't have much experience with extracellular vesicles, but I think it is unlikely that antibody-bound vesicles exist which you would remove via the Protein G.
Carsten
Thank you all for your answers! I will probably try to biotinylate the antibodies.The secondary antibodies I am using are already supposed not to detect denatured/reduced IgG, but this is not working very well and still detecting the IgG of the lysate.
Hi,
You may want to try a secondary antibody that does not bind to denatured IgG. We use Trueblot from Rockland and it works pretty well.
http://www.rockland-inc.com/trueblot.aspx
-Xiwei
Dear Xiwei, this is the secondary antibody that I am using, but still, it recognizes the denatured/reduced Ig...
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