Does anyone have a protocol for slab-IEF for minigels (1.5 mm thick, 7cm)?
After IEF I would like to transfer the proteins to membrane and do immunoblotting (without second dimension run). The pI of the protein isoforms of my interest is 4-5.
IEF gel, 5%; 1x (7.5ml)
40%T/3.3%C (acrylamide/Bis) 937.5 ul
80% glycerol (5% final) 468.8 ul
Ampholyte (pH 3-10) (40% from Sigma) 375 ul
TEMED 3 ul
Water 5.7 ml
degas
0.1% (w/v) FMN 50 µl (FMN: riboflavin-5'-phosphate, can use riboflavin instead. )
10% Ammonium persulfate 20 ul
Total 7.5 ml
Leave under light >8 hours after pouring.
Lower buffer chamber (IEF Anode Buffer): 7 mM phosphoric acid
Upper buffer chamber (IEF Cathode Buffer, pH 3-10):
20 mM lysine, 20 mM arginine, pH10
2X sample buffer: 40 mM lysine (free base); 40 mM arginine (free base); 30% glycerol.
Load 5ul protein + 5 ul 2X sample buffer + 0.15 ul 0.1% bromophenol blue, without heating.
Running conditions: 100 V constant-1 hour; 200 V constant-1 hour; 500 V constant-30 min.
Fixing gel: fix in 12% TCA for 30 min.
Stain as usual.
After IEF, proteins on the gel are all at their PI and neutral. Hence transferring to membrane with electroblotting will not work. The old method using tissue paper should work.
IEF gel, 5%; 1x (7.5ml)
40%T/3.3%C (acrylamide/Bis) 937.5 ul
80% glycerol (5% final) 468.8 ul
Ampholyte (pH 3-10) (40% from Sigma) 375 ul
TEMED 3 ul
Water 5.7 ml
degas
0.1% (w/v) FMN 50 µl (FMN: riboflavin-5'-phosphate, can use riboflavin instead. )
10% Ammonium persulfate 20 ul
Total 7.5 ml
Leave under light >8 hours after pouring.
Lower buffer chamber (IEF Anode Buffer): 7 mM phosphoric acid
Upper buffer chamber (IEF Cathode Buffer, pH 3-10):
20 mM lysine, 20 mM arginine, pH10
2X sample buffer: 40 mM lysine (free base); 40 mM arginine (free base); 30% glycerol.
Load 5ul protein + 5 ul 2X sample buffer + 0.15 ul 0.1% bromophenol blue, without heating.
Running conditions: 100 V constant-1 hour; 200 V constant-1 hour; 500 V constant-30 min.
Fixing gel: fix in 12% TCA for 30 min.
Stain as usual.
After IEF, proteins on the gel are all at their PI and neutral. Hence transferring to membrane with electroblotting will not work. The old method using tissue paper should work.
Thank you!
About the electroblotting.. I have used Immobiline DryStrips for IEF after which I have run them on SDS-PAGE gels and transferred to membranes with semi-dry electroblotting. Isn't this the same thing as with slab-IEF gels, that the proteins are still neutral, but are still transferred?
After SDS-PAGE, the protein molecules are coated with negatively charged SDS molecules. I guess one can also soak an IEF gel extensively to wash out the Ampholyte before subjecting it to electrobloting.
I successfully electroblotted proteins from slab IEF gels by equilibrating and tranferring in 0.7% acetic acid, transfer time 1h (places of membrane and gel were reversed because proteins will be positively charged with acetic acid). Worked really well.
Hi Sheila, yes it is possible to transfer proteins from slab-IEF to a membrane and do immunodetection. However, I do not know what could be the cause of observing a smear instead of a band. Maybe there's too much salts in the sample buffer? Or if the gel gets too heated during the run. I use cold running buffers, and do the run in cold room, and put the running equipment to a water-ice bath.
Hi Mervi. Will this method of blotting with 0.7% acetic acid work with PVDF membranes? Do you prewet the PVDF in methanol as usual and then wet in 0.7% acetic acid, before blotting?
Yes, I'm using Millipore-FL PVDF membranes.
The membranes must be always prewetted in MeOH to be "activated". Then briefly in acetic acid.
Hi fellows, I'm trying to perform an IEF in slab gel. About the protocol presented here, is the FMN essential? What is the role of FMN? I'm asking because I don't have it in my lab. Another question: If I want to denature my proteins, can I add urea to this mixture?
Thanks in advance
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