Sometimes I prepare a standard SDS-PAGE gel for westerns, wrap with a wet paper towel and cling wrap, and store it in a zip lock baggy in the fridge. Does storage for a few days alter the effectiveness of the gel in terms of sample separation, transfer, or background on the final image?
I know that our proteomics core claims that SDS-PAGE gels used for mass spec should be freshly made. Has anyone ever tested this for westerns?
If you want to store the SDS-PAGE gel overnight at 4C, you should not pour the staking gel (pH6.8) over the resolving gel (pH 8.8) as the pH gradient at the interphase between these gels would change over time due to diffusion. Hence I would suggest that you pour resolving gel, overlay with 1X pH8.8 Tris buffer, store at 4C, which can be stored for several days. on the day of your run, get the get to room temperature, pour the staking gel to proceed further.
I never had a problem using a gel stored about for 2 weeks (I didnt intentionally test for longer duration) for western blots.
If you store it in the way you described it, you can at least store it for 2-3 weeks. I have done that without problems.
I normally soak the gel in the SDS running buffer using a paper towel & store at 4 degree C. If you have observed, even the commercially available gels are packed in such buffers. These commercial ready-made gels have long shelf-life and I guess they do not differ much from home-made gels.
Have tried storing SDS-PAGE gels for a few days like what you mentioned. Wrap gel with moist paer towel followed by a cling wrap and store at 4 C. If overnight, layer top of gel with water and cover with cling wrap and leave in fridge.
If you prepare the gel w/o SDS it should be fine for months..Just that you need to soak or equillibriate it for sometime in running buffer..Also SDS travels faster than your protein so by the time the protein reaches particular region the environment is already denatured....
Most of the commercially prepare gel use the same principle if I am not wrong...
I know people using this same technique very successfully
If you want to store the SDS-PAGE gel overnight at 4C, you should not pour the staking gel (pH6.8) over the resolving gel (pH 8.8) as the pH gradient at the interphase between these gels would change over time due to diffusion. Hence I would suggest that you pour resolving gel, overlay with 1X pH8.8 Tris buffer, store at 4C, which can be stored for several days. on the day of your run, get the get to room temperature, pour the staking gel to proceed further.
Definitely for Mass-spec you don't want to take chances, but for daily usage at least for a week or two they work fine when stored like that. Just for comparison I think the tris-glycine precast gels have 3-6month shelf life (other buffer systems more common now like bis-tris have around 1 year)
To store the gels (under optimal conditions) not longer than 7 days will not significantly negative affect your work
As Moorthy mentioned above, gels without stacking can be stored really for weeks or months at 4 degrees. We normally make gradient gels (separating gel only) in bulk and store it immersed in separating gel buffer with SDS.
Complete gels can be stored in the way u mention for a few days if the wells and the stacking separating boundary looks ok (not dried / cracking)
I gave it a try with a gel I stored the way you describe and then left by accident for ~6 months in the fridge: it worked fine for sample separation and coomassie staining. but I did not test western blotting.
You already got pretty good answers and I do not want to hammer the nail any more. I would like to add that one of the reasons it is not recommended to store home-made gels for longer than two to three weeks (even at 4C) is that the stacking gel will slowly start mixing up with the separating gel and hence disrupt the interface between the two. The longer you store them, more damage is done to the interface and the samples will not stack properly before they start migrating on the separating gel. That is why, as others suggested, we usually pour the resolving gel and store it and add the stacking gel when we want to run it.
Normally SDS precipitates in cold, so storing a gel in 4C overtime will lead to SDS precipitation and affect protein seperation. Thats why commercial gels have no SDS in them. Taking this into consideration, its best to use fresh gels, but if you have to store them dont store for more than 2 days. Incidently our proteomics facility always sugests BIO-Rad precast gels as fresh gels always have some amount of unpolymerised Acrylamides that affects results.
If you prepare gel as usual way you can store upto 10 days. If you want to store longer time means 1. avoid SDS in the resolving gel, 2. don't cast stacking gel and 3. cast the gel at 4 degree gel. It will reduce the contraction of your gel. Likewise if you process your sample at 4 degrees you can get better result for such a case.
For western we use cling wrap to support the gel and we place the gel directly onto cling wrap. Use a second sheet of cling wrap over the gel and wrap the cling wrap around the gel and keep it in a -70 freezer.like this we ve been able to keep the gel for some days (we did not try more. Maybe it works!!)
hi ,
If you need to store your gels , only 1 or 2 days is ok in 4 degree . But i like to use fresh gels. Results are very different . By the way Aparna Mukhopadhyay 's very right. Good luck :)
hi, as long as the gel (especialy the wells) is full with ddw you can save it in 4C for 3-4 days
I have stored gels the way you described wrapped in wet paper towel (wet with running buffer) and in a cling wrap @ 4˚C for 1 week and successfully used it. I notice these gels are more brittle than if I made them fresh. I have been told that pH changes with longer storage. I haven't stored for longer than a week. However, the pre-cast gels from companies are stored for months without any adverse effects. They also store it with running gel (they do add sodium azide though).
I would say that pH changes are real because when you use old running buffer the gels run very weirdly (even freshly made gels).
If u store it at 4 degrees then u cn use fr a week or so.. Wrap in a plastic bag... N do check it often
A uniform pore size SDS gel can be stored for 2-3 weeks at 4 degrees. If you have a gradient SDS gel say, 4-20%, I have stored them for up to 2 weeks with no noticeable change in the results obtained. The only precaution to be taken is to make sure the gel has solidified well before you put it in the cold room, and not to have added the stacking gel to the separating gel. Keep the gel well layered with water/ water+butanol mix, and replenish this every few days to prevent complete evaporation, as this will cause your gel to shrink and become unusable.
It is always recommended to use freshly prepared gel. If delay is anticipated store at 4 C no longer than 16 hrs for best result.
In our lab we usually make gels before the day we run it and store it in 4 degree C with a wet paper sealed with aluminium foils. If in case we don't run the gels the within 2 days, we usually discard it not to waste time in taking risks. Please make note, if the samples that you are going load are in very less quantity and from precious tissues or cell culture then I highly deco you not take any risk as you lose all your sample and eventually ending up with waste of materials and our precious time. So it is better to make fresh gels. On the other hand make yourself a time limit for the storage of gels (in our lab not more than 2 days), as you vary each time the storage time though the running is good but you cannot reproduce the results. I hope this comment helps. Good luck with your WBs.
Hi e1 ! Need some good advice
i prepare 12% sds gels for my rice leaves proteins but i am facing problems in running the gel , it takes 16 hrs to run completely at 150 v , 50ma and 12 w. Y is this so ..i am following laemili running buffer composition and evey dilution is well prepared. Help plz !
@Kamran: I would recommend opening a new question for your question since its not related to the topic here and the visibility is also better.
Hello Brad!
If you don't need the gels for mass spec you can prepare the gels and store them for a while.
I routinely prepare my gels (upper and lower) the day before use and store them after polimerization in the cold room leaving the comb inside the gel to preserve the wells.
If you don't need the gel soon you can store them as you described up to one week without any problem but I recommend to use a cold room instead of a fridge. I did it many times without any problems in separation and transfer of proteins up to 147 KDa.
Hope this helps.
MG
Dhatchana Moorthy gave you the best answer for your query. goods luck
Exciting replies interesting, I agree with most of the answers above.
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