We have attempted western blot for detecting some low molecular weight proteins and I have a problem with running my exosome samples and after probing I get wavy bands but not the cell lysate samples. The exosome sample is running slower in 10% gel compared to the lysate samples. Any thoughts please?
It sounds like the exosome samples are in a high salt buffer. If the salt concentration is too high the samples will run slower and produce wavy bands, they can even distort the bands in the next lane. You may need to dialyze or desalt you exosome samples. A number of mini dialysis devices are available, such as the "Slide-A-Lyzer." Be sure to choose a device with a low molecular weight cut off to prevent losing any of your low molecular weight proteins.
Hello Alan. Thanks for your answer. We looked at that possibility too. However we processed both exosomes and cell lysates in same buffer, but why cell lysate samples did not show any retardation in the gel compared to exosomes? Herewith attached our gel and blots of the same experiment. We see a different migration pattern for exosomes and cell lysates in the gel. and this is not the first time! I am puzzled...!
Hi Anish, I agree with Alan that in the process you may be retaining or maybe creating new salt complexes. However, here is my thought. I have always isolated extracellular vesicles from media by centrifugation but I have never isolated them using RIPA. Instead, I have resuspended them in PBS to check protein concentration (no need for protein inhibitors since other than MMPs there are no proteases in vesicles, just keep in cold) and then resuspended them in loading buffer to follow by boiling. Sometimes the pellet can be resuspended directly into the loading buffer just to check presence of specific marker. Try ommiting the RIPA step and see how it goes. I hope this helps.
best luck,
edgardo
Hi Anish, It also could come from a big amount of BSA which is contaminated your prep and delaying running. Did you do a ponceau staining ? if you see a big bad around 80kDa, it could be that but I dont have a solution for that! Good luck, Karine.
Hi Karine..There is no source for BSA contamination here. Anyways thanks for your response.
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