Hello,
I am planning to perform proteomics on exosomes as part of my project. Previously, when we had performed proteomics on few cases we only observed an abundant amount of serum proteins and I could not locate any exosome relevant proteins. I was told that depletion of abundant proteins like albumin and IgG would help in detecting low abundance proteins. Following this, I used Pierce top 14 depletion column to pass my isolated exosomes and submitted to the core facility. They informed me that they could observe a resin carryover in my depleted samples and will not be able to run them on the gel.
Could someone suggest what the possible issue could be? I am sure I followed the entire protocol mentioned for depletion using the columns as is. Any suggestions would be very helpful!
Thanks,
Hi Sneha Garudeswaran
What format of the depletion was used (loose resin or spin-column format). What do you mean by resin carry-over; are you getting the immobilized antibody peptides...
If you are using Ultra-centrifuge based exosome isolation; you should not observe any serum proteins; therefore depletion step is not required...
Consider using Plasma rather than Serum for isolating Exosomes; we have faced issues in the past while using Serum for Exosome isolation.
@ Saravanan Kumar,
I used the High-Select top-14 abundant protein depletion spin columns for depleting my samples of high abundant proteins. I isolated my exosomes using UC and ultrafiltration approaches and I still find only serum proteins and no exosome related proteins, which is why I used a depletion column to get rid of the serum proteins. The person at the core facility informed me that he found the sample to be gelatinous and could identify gel beads in my depleted sample, which indicated the possible presence of antibody. He said presence of antibody in the sample would not help in performing proteomic analysis of these exosomes. Is there anything that you could suggest so that I will be able to optimize my sample preparation for proteomics?
Thanks,
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