In my memory, you should first do  isoelectric focusing, and then do SDS.

Do you solubilise your protein extract (to be run in IEF) in rehydration buffer (urea/thiourea, CHAPS) containing carrier ampholyte and DTT/deStreak? Proteins solubilised in SDS buffer/Laemmli buffer usually are not performed well in IEF as SDS will interfere the focusing. SDS will coat the proteins to become more negative charge. Therefore, it is recommended to solubilise your protein in Urea rehydration buffer.

Agarose will not affect the protein migration. In fact, besides holding tightly on your IPG strips, it should allow all focussed proteins to migrate into SDS-PAGE gel due to its larger pore size. During equilibration step, do not agitate/shake vigorously, just gently will do. Equilibration step is important to ensure the successful of the subsequent separation on SDS-PAGE. Proteins are negatively charged in this stage, thus, they are easily to be separated (negative to positive). IPG strips immerse in running buffer for a while should be fine. I usually do this for less than 1 min.

You need to equilibrate the IPG strips after IEF for longer than 1 minute. I think the usual procedure is 15 min in reducing/SDS-containing buffer followed by 15 min in alkylating/SDS buffer. You could also do a test run of the IPG/IEF stage and visualize the protein on the IPG strip with Coomassie Brilliant Blue to make sure your sample entered the first dimension and focussed.

@Boon Chin Tan: thanks for that suggestion and ofcourse I did the solubilisation of proteins in rehydration buffer only. Planning to silver stain the gel and will look whether its due to the problem of low protein load.

@Bruce Allen: thanks for that suggestion. Even we also did the equlibration for 15 minutes each and after that dipped the IPG strip in a cylinder with the 1x TGS running buffer. Thought of doing the staining of IPG strip, but since the strip is little bit expensive we just skipped that step and directly proceeded to the second dimension. I am wondering whether urea does any modification of those proteins that may affect the quality? or the removal of salts made the protein to precipitate?

thank you all for your valuable suggestions !!!!

Here some procedures that guarantee a succesful separation by 2DE.

The same as Hong Yu, i think the problem is in your IEF. Stain your IPG strip. Maybe you loose your proteins during this procedure? for equilibration I use DTT and iodoacetamide.

Muralidharan, I am glad you bumped up equilibration to 15+15' (reduction and alkylation).  There are inexpensive trays (rather than tubes) for that so you can make sure that the equilibration happens homogeneously. Post-stain the IEF strips after the run to see how much protein was left behind. First off, how much protein do you load? If there is too much proteins within the same pI range they may precipitate in the strip and not transfer to the 2nd D. That is, more protein does not always transfer quantitatively to the second dimension and you  have to find the right balance for your samples: load vs. transfer vs. resolution. Also, what type of samples are you separating? If there's a lot of membrane proteins you may have to bump up the SDS % both in the equilibration buffer and the running buffer. I normally don't add SDS to the gel mix to prevent bubbles but that in the running buffer will equilibrate immediately. Just to give you an example for mitochondria preps you can go as high as 9% SDS for basic proteins (6-11). The problem then is with MS. In case you use a lot of SDS in the second dimension you have to fix the gels at least overnight, and rinse in bidistilled water thoroughly. This is to avoid carry over of SDS to the MS. Hope it helps and good luck!

We already observes the same think even if we respected protocols from proteins extraction to sds page migration. The problem came from your first dimension and in our case the problem came from the strips themselves. Sometimes, When we opened the plastic film from the strip the gel broke at different position. Do you observe something like this ? And for other users ? I ask myself about the way or Time to wait after pick off strips from -20 c ?

Recheck with your every step of IEF, answering your question about overlaying agarose surely it will not have any impact on the run( since i am sure you would not overlayed agarose which is boiling hot. Check with your staining procedure if you want please mail me your the staining procedure you used i can suggest you further on it and i can also mail you the procedure which i use in my lab.

@Giulio Agnetti : Thank you very much for the suggestion. Infact i have loaded 200ug of protein on to the strip after  methanol: chloroform precipitation. We did this so as to remove the salts,lipid contaminants from the sample and have centrifuged the samples before loading on to the strip. Interestingly when I did silver staining of the same strip I got few spots with streaking in it (please find the attached gel image). I am wondering what could be the reason for this kind of gel? and one thing which is more confusing is that eventhough I have loaded 200ug of proteins coomassie is not giving any result. Why the spots are not visible even-though the detection limit of coomassie is 100ng??? does this means I have to load more protein samples? and If i do so will it affect the IEF focussing in the fiorst dimension in any way??

FYI: The protein sample was prepared from a cell line. Infact we suspect that it is a total cell lysate !!!!! A gel loaded with total protein showing only few spots !!!

Once again thank you very much

@Ingrid Fruitier-Arnaudin & Pavel Májek: No we didnt faced any problem like that and as Pavel Májek suggested we thawed the strips in the room temperature for 1-2 minutes and then proceeded with the rehydration step.

Thank you very much,

@Usha Talambedu : thank you very much. Happy to share with you the protocol that we used in our lab. Please provide me your mail ID ([email protected]).

Thank you very much,

Is that image a silver stained 2D gel in which you load the 200ug of proteins? If that is the case I suppose that the problem is in the loading amount or in the protein amount estimation. Which is the length of immobiline that are you using?  With sort 7-8 cm immobiline you get a lower number of spots. It also seems that you still have problems with salts. Try to was better your sample after precipitation and try to change the electrodes papers during the run when the cathode paper became yellow.

@Roberto Raggiaschi: yes this is the silver stained image of the gel with 200ug of protein in it. We have used 7cm  pH3-10 IPG strip and wondering what could be the problem with the spot detection. We have rechecked the protein estimation but couldn;t find any mistakes there too. We have precipitated the samples prior to rehydratiion to remove salts and other interfering substances using methanol:chloroform precipitation. I am just wondering weather methanol:chloroform or TCA/acetone works well with this. We are sure that there are no salts in the samples because the current in the gel was limited only to 25uA during focusing and the desired set  voltage is reaching with the provided time. Will nucleic acids also precipitate with methanol:chloroform technique. Please provide me with your suggestions.

Thanks,

MDV