Hi there,
Once frozen you can store your samples for a long time. I would suggest to boil them again prior loading.
Since you have denatured your samples, I think the sample can be shtored for a long time even at room temperature.
you can store samples at 4 degree in a fridge, preferably for overnight only.
Agreed to Mr Vivek. Samples with SDS should not be freezed. But you do you want to store the samples. Store the protein instead and prepare the final solution only when the gel is ready.....
As per my experience, I have not stored my samples after mixed with dye, because the dye may damage the proteins during low temp. And I got differences from fresh/ stored same samples.
From my experience it is fine to store denaturated samples for few hours in room temperature, up to 24 hours (maybe a bit longer) in -4 Celcius degree.
After that time sample may not be as clearly visible on the gel so it is better to perform electrophoresis asap.
On occasion I used sample stored in freezer (-20) for few weeks, but like mentioned above, you have to boil the sample again.
I agree with Aniruddha. Take into consideration that proteases continue work slowly at 4*C in presence of SDS and denatured proteins are better substrate for these enzymes than natural ones.
I did stored the denatured samples at -20 degree and used it after some weeks which worked well for me.
I also stored my samples in sample buffer (unboiled) at -20°C for days/weeks without any problems. Since they were radioactively labelled in most cases I tried to run them within a few days.
When I wanted to run them I boiled and vortexed them, which will also dissolve SDS precipitates.
Store at -20C or -80C, I've been doing it for years, they still are good. When you need to run them, thaw them and give them a quick spin (I never had to boil again after taking out from the freezer, thawing at room temp should take care of the SDS precipitates, if not then you can boil it for 1-2 mins)
We have an assay for a protease where the reaction could be stopped only by addition of Laemmli sample buffer plus freezing at -20 deg C right away. The enzyme would continue working otherwise. Since we stopped the reaction at different hours, we could not add the Laemmli sample buffer to all the samples just before running the gel. However, we would not do the 5 min 95 deg C treatment on the heat block until just before running the gels.
In general, you should be able to store them indefinitely at room temperature, as they have been denatured by boiling in loading buffer. This works for plasma proteins, for example. We did this routinely for prac classes when I was at a former university. In some cases you might have to increase the concentration of SDS.
Since the protein has already been denatured, you can store indefinitely at such temperatures as can prevent growth of microorganisms and hence possible degradation of the protein. If you are trying to keep for a long time, storing at -20 degree Celsius should be appropriate. But when ready to use, thaw gently to avoid further denaturi,g your protein.
It is safe to store the samples in the loading biffer after boiling overnight or a day at 4 degrees C. But it is always preferable to prepare the samples fresh, as some residual protease activity still might be there, so that storage for a long period of time is not recommended, even though in many cases, it will probably be ok.
How long could the sample buffer be stored? Or should it be prepared fresh all time?
How long can I store a SDS-PAGE loading sample (protein) after it's being mixed with dye and denatured (boiled for 5 min)?
Samples can be stored for few months at -20 C for longer storage they can be stored at - 80 C.....
Regards
Another thing you may want to consider: SDS-PAGE preparation often involves use of an alkylation agent like iodoacetamide to prevent disulfide bond reshuffling after denaturation. Disulfides tend to break and re-form in solution (there is an equilibrium). In the presence of an alkylation agent they can't re-form , so you could see disulfide bonds start falling apart over time.
- Badges
- Science method