Hello, All,
I am working on exosomes currently,and I have been using whole cell lysates as positive control. I always treat my cell lysates with enough amountg of RIPA buffer, estimate the concentration and take the required amount and store the rest in -80 degrees. Can i reuse these cell lysates when I run the western blot again or do I need to prepare fresh cell lysate each time? I have been having issues with western blots of late, and prepping the samples each time is really getting tedious. Suggestions or answers would be greatly appreciated.
Thanks,
Hi there
- you can definitely re use
- i would add that for immediate use over 1-2 months keep your lysate at -20C rather than -80C as repeated freeze thawing from -20C is less deleterious to proteins
- i woukd also add that you should thaw your lysate s gradually on ice over 30 min and try not thaw more than 3-4 x
- Thus make smaller aliquots archive at -80C for up to 1 year
- Transfer single aliiquots to -20C for immediate use over 1-2 months and freeze thaw up to 3-4 x.Discard what remains - hence the need for smaller aliquots - and then move onto the next archived aliquot
- I should finish that the above imperatives are particularly applicable to quantitative protein work
Hi there
- you can definitely re use
- i would add that for immediate use over 1-2 months keep your lysate at -20C rather than -80C as repeated freeze thawing from -20C is less deleterious to proteins
- i woukd also add that you should thaw your lysate s gradually on ice over 30 min and try not thaw more than 3-4 x
- Thus make smaller aliquots archive at -80C for up to 1 year
- Transfer single aliiquots to -20C for immediate use over 1-2 months and freeze thaw up to 3-4 x.Discard what remains - hence the need for smaller aliquots - and then move onto the next archived aliquot
- I should finish that the above imperatives are particularly applicable to quantitative protein work
Thank you very much for your suggestion. I was feeling it very tedious to prep my samples all the time, since I had a lot of controls to use for my validation process. And, yes this time I will try aliquoting them and store at -20 degrees and the rest at -80. Thank you once again!
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
Sure. You can reuse them many times. The only thing is that afterI used to do it very often. freezing-thawing some aggregates may appear (althiugh unlikely in SDS which is present in RIPA). oprotein Another thing is that if the samples were stord with Laemmli sample buffer, frequent heating at 95oC may lead to fragmentation of some proteins. So usually I heat at 95oC only once. Then, every consequent use I heat the samples at 50oC.
I should also add to my original answer by reminding you to add protease and phosphatase inhibitors to your RIPA buffer to ensure that your proteins do not degrade during storage and freezethawing as I have already described
Thank you very much for all your answers and suggestions. And, Dr. Laurence, yes I do add protease and phosphatase inhibitors every time when I prepare RIPA buffer.
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