SDS-PAGE gel preparation - (Jun/28/2006 )
hi guys
i have a question
i read that its ok to make the gel and leave it even overnight ...... how to leave it? i mean do u wrap ur gel inside the glass slides in the refrigerator or do u put it in the buffer ,the running buffer ,i mean till sample loading?
one of collegues said we have to prepare the gel immediately before loading the sample or the protein will migrate diiferently ?????
thankx again
i have a question
i read that its ok to make the gel and leave it even overnight ...... how to leave it? i mean do u wrap ur gel inside the glass slides in the refrigerator or do u put it in the buffer ,the running buffer ,i mean till sample loading?
one of collegues said we have to prepare the gel immediately before loading the sample or the protein will migrate diiferently ?????
thankx again
It is OK to wrap the gel inside the glass plates with cling wrap, then leave it in cool room @ 4 degree, and use it tomorrow.
The difference between leaving the gel to set overnight and use immediately:
1. the gel will be fully polymerized if left overnight,
2. the stacking gel will shrink more if left overnight, therefore the well may become shallower.
3. bubble may develop between the gel and the plate due to shrinkage, don't worry it will not affect the sample running in the gel....as the bubble does not exist inside the gel...
4. do not leave the gel for more than two days.
I hope this may help.
Minnie Mouse
U ARE A LIFE SAVER really thankxxxxxxx
I leave gel overnight in casting apparatus, wraped in sealable plastic bag to prevent evaporation of water layer put on top of gel (bottom of gel is sealed by rubber seal of casting apparatus). I do not cast stacking gel, only resolving - then polymerize stacking for one hour next day, while preparing samples.
As good as I know overnight polymerization gives more reproductible results.
At my lab, we use a ton of gels all the time (3 or 4 a day). We tend to make a bunch at once and store them all by washing and filling wells with water and then wrapping the gel in wet paper towel before placing in a Ziploc bag (with a little water inside so the towels don't dry) at 4 degrees. These are 12.5% gels and keep for a few days no problem.
We used to keep gels wrapped in wet paper towels and leave it in 4C. Now we just buy them. 
thankx guys
though my mentor isnot convinced, he told me to try it bymyself so i am gonna prepare 2 sets and try ur way and let him know the results.
thankx
what is the function of stacking gels? n what is the mechanism of protein separation by SDS-PAGE?
The stacking gel is a low concentration polyacrylamide gel with a pH that is lower than the pH of the running buffer. With this low pH, the glycine ions become zwitterions (charge zero) that will be able to enter but they will not be able to run through the stacking gel. Since the number of charged ions in the stacking decreases when glycine becomes a zwitterion, the voltage in the stacking will increase and therefore the proteins will run very fast through the stacking and will compact on the front of the separating gel. Here, the pH is higher and, when the glycine eventually reaches the separating, it will restore the voltage in the stacking but it won't change it in the separating. In the separating the proteins will be separated according to their size
please read this
this is the most intersting site i read about SDS PAGE
http://mullinslab.ucsf.edu/protocols/html/...GE_protocol.htm