Proteins and markers in top of gel - Running a gel (Jul/04/2006 )
Dear all,
I am trying to seperate out plant proteins by following a protocol by Saravanan and Rose (Proteomics 2004) However I only seem to get the proteins into the gel and then they only run about a quarter of the way if at all. This is also the same for my commercial prestained markers (BioRad)
I submit the steps that I have been taking below. I have also attached jpegs of some examples of gels if anyone would care to look.
If any one has suggestions I would be most grateful.
Phenol Extraction Method (after Saravanan & Rose 2004)
5 g of frozen plant tissue was finely powdered in liquid nitrogen using a pestle and mortar and resuspended in 15 mL of extraction buffer (Extraction Buffer :1% (PVPP), 0.7 M sucrose, 0.1 M KCl, , 0.5 M Tris-HCl pH 7.5 , 50 mM EDTA , 1mM (PMSF), 2%-mercaptoethanol )
The mixture was extensively homogenized on ice. Then an equal volume of Tris-HCl pH 7.5-saturated Phe was added and the mixture was rehomogenized for 30 min on ice. Centrifuge at 4,500x g for 45 mins at 4OC
Remove the upper Phe phase and re-extract 2/3 times with extraction buffer
Proteins are precipitated from the final Phe phase with 5 volumes of saturated ammonium acetate in methanol overnight at –20OC and finally centrifuged at 4500 x g for 15 mins.
Do a Bradford assay to estimate protein concentration and standardise loading amounts.
Wash protein pellet in ice-cold methanol followed by several ice-cold acetone washes.
Dry out the tubes in the laminar flow cabinet.
IPG (17cm pH 4-7) strips were re-hydrated overnight (gels side down) with 300µl of IEF buffer (7M Urea, 2M thiourea, 4%chaps, 10mM DTT, 1% w/v carrier ampholytes (pH4-7)) Our protein concentrations were between 0.5 and 1.2 mg/ml.
Strips were focused (place a purified water-moistened wick under the gel and over the wire of the electrodes) at 20 oC in a focusing tray using a linear increase from 0-500V over 1 h, 500- 10,000V over 5h and held at 10,000V for a total of 100kVh.
12 % Gels were cast using the Ettan Daltsix gel casting system. Rig and plates cooled overnight to 4 degrees. Gel ingredients minus temed and APS were cooled on ice for 3-4 hours and then polymerization ingredients were added and gels poured. They were allowed to polymerize for about 2 -3 hours at room temperature water saturated butanol was over laid on the gels and removed prior to storage in the fridge overnight.
IPG strips were capped (1% w/v DTT in Equiblibriation buffer) and alkylated (2.5 % w/v iodoacetamide in Equiblibriation buffer (6M Urea, 30%w/v glycerol, 2% SDS,
50 mM Tris HCl pH8.8) the strip by transferring to rehydration trays (gel side up) and adding the above solutions (~5ml per gel) in sequence with purified water wash in between.
Align the focused IPG strip with the plastic coated back to the larger plate on top of the 2nd dimension gel. Add wicks with 10µl of comassie stained markers on either side of the IPG strip. Seal with warmed agarose solution.
Anodic buffer was as follows: 250mM Tris, 1.92M glycineand 1% SDS
Cathodic buffer had 2x SDS concentration of the anodic buffer.
Gels were run on the Ettandaltsix system. Initial runs were done applying 100v over 16 hours (from Saravanan and Rose)
Recent gels were run at 5w per gel for 30 mins and 17w per gel for 5 hours (from manufacturers manual).
In all cases the proteins don’t seem to run fully down the gel and neither do the markers that we are using (BioRad). We have been repeating this over several months changing suppliers and tweaking it but we still get the same result! 
So if anyone can think what the porblem might be I woudl be most grateful
Regards
Raf-Mc
Maybe you already know it but, please, have a look at this:
http://www.ruf.rice.edu/~bioslabs/studies/...e/sdsgoofs.html
I don't think it could be the sample preparation because your marker runs strange... The running buffer is the same we use. I think it could be as they show in the website I mentioned above: maybe you have a problem with acrylamide. Did you buy a new bottle? Could it be a different percentage or a different ratio acrylamide:bisacrylamide? It's just that everything looks fine to me...
Cheers for that site. Good to know that we all make mistakes 
However I doubt as suggested there that we ran for too little time. 4.5 and 16 hours isn't a short run!
Thanks anyway 
However I doubt as suggested there that we ran for too little time. 4.5 and 16 hours isn't a short run!
Thanks anyway
Actually, something looking like your gel was not supposed to have run too little. They said it could be due to the wrong percentage of acrylamide. Did you try to use precast gels? Just to exclude the possibility...
Actually, something looking like your gel was not supposed to have run too little. They said it could be due to the wrong percentage of acrylamide. Did you try to use precast gels? Just to exclude the possibility...
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Yes I think I will try precast though all of our acrylamide was newly bought from Sigma and I thought that 12% woudl be fairly standard. Still it is worth a try to change it about a bit.
Thanks
S