From the image you provided it seems you are having a problem with sample preparation. You may want to consider a few options.

1. Precipitate your protein sample first and then re-solubilize in the focusing buffer

2. Treat the sample with a little benzonase to remove DNA contamination.

3. Be sure that your focusing buffer is prepared correctly with urea (6-7M), thiourea (2M), chaps (2-4%), reducing agent DDT (100mM), and the IPG buffer at 1-2% (ampholytes) recommended for the strip. All of these components are necessary for good focusing.

Thanks for suggestion

Dear Dr. Carroll

As per your suggestion, We have treated the protein sample with DNAse and RNAse in order to remove nucleic acid contamination.

The sample has been  re-precipitated with 100 % TCA and then resolubilized in our focusing buffer.

Our  focusing buffer is from GE Health Care which contains 6M urea, 2M thio urea, 2% CHAPS and DTT of 100 mM concentration.

The IPG strip used was 7cm , pH 3-10. The isoelectric focusing conditions were initial rehydration at 20 deg celsius at 150 V for 2 hrs, 300 V for 1 hr, gradient 1000 v for 2 hrs., gradient 5000 V for 2 hrs., and final hold at 5000V.

But after following all these conditions, we are unable to resolve our proteins as separate spots. 

Our protein sample was initially solubilized in 5% SDS containing 0.1N NaOH. Will TCA precipitation  remove these contaminants. Secondly, we used 2D clean up kit (GE Health care) for the removal of non protein contaminants.

Do we have to further modify our sample preparation ? 

What is the significance of higher strip lenght (18 cm or 24 cm ) in protein separation.

The latest 2D gel image is attached.

Regards

Divya 

Attached is the protocol that I have developed for analyzing samples from mouse tissues, Borrelia burgdorferi, Tularemia, and Leptospira. See how this compares with your procedure.

I typically use 11 to 18 cm IPG strips and focus anywhere from 80 to 200 ug of protein per IPG strip.  With the pI 3-10 strips, the resolution is typically only pI 4 to 8.5. I have found that one will rarely separate proteins below or above these pIs as they tend to either stack at the electrode ends or migrate off the strip.

I don’t use TCA precipitation anymore, but typically perform the Methanol/Chloroform/Water method (Wessel and Flugge. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry. Volume 138, Issue 1, Pages 141–143). I get better protein recovery and better solubilization in my focusing buffer.

There are advantages to using the longer strips as you can load more protein and sometimes obtain a wider distance between distinct spots. With the 7 cm strips you can overload them resulting in poor focusing and the spots can be very tight depending on the spot pattern.

Also, I use an IPGphor Isoelectric Focusing System... not a Multiphore II system. Not sure which you are using. They are different and require different focusing parameters. From parameters you have provided it would seem you are using the IPGphor system??

Another issue is time of sample solubilization and protein amount per volume buffer. I typically incubate my protein samples in the focusing buffer at room temperature with rotation or gentle shaking for at least 2 hours prior to application to the IPG strip. Many proteins will take time to go into solution especially after precipitation, so you must allow ample time for this to occur. The concentration is typically 1ug per ul buffer, and then we dilute the stock sample with additional focusing buffer to bring to the appropriate volume depending on the strip length. 

I'll end with this, I incorporate the sample into the rehydration step of my protocol (see attached), where we rehydrate the strip with sample for 12 hours before focusing. Making sure the strip is fully hydrated is also critical to good focusing.

Dear Dr. Carroll

Thank You so much for the methanol precipitation protocol suggested, so that we could successfully generate distinct protein spots.

But now we are in another trouble. After methanol precipitation, the protein spots are of too low intensity, so that it is barely visible.

What could be the reason of low intensity after methanol precipitation? I it due to protein loss during purification?

Is there any suggestion to increase intensity?

Latest gel Image attached herewith.

Regards

Divya Jose

Dear Dr. Carroll

Also we have high back ground staining on gel image. We perform silver staining using proteoPlus Silver stain kit from Sigma.

If you are losing your sample using the Methanol/Chloroform precipitation method you may want to reconsider using it are return to your previous method of TCA. Go with the procedure that works for you.

Loss of recovery using any precipitation method can occur if the protein pellet, which is often difficult to see in the bottom of the tube, is dislodged when the liquids are removed. Not sure of the volumes you are using, but I typically use a hand pipet to remove the liquid from the tube and try to keep an eye on the protein pellet so it is not disturbed.

Just so we are on the same page about the Methanol/Chloroform procedure, below is my simplified version that my lab uses:

Methanol/Chloroform/Water Precipitation of Proteins

1.            To protein add 4X volume MeOH (vortex)

                then 1 to 2X volume Chloroform (vortex)

2.            Phase separation

                Add 3X volume dWater (vortex)

                Spin 1 min at 12,000 x g

                Discard upper (aqueous) phase (be sure to not disrupt the protein layer at the interphase)

3.            Add 3X volume MeOH to lower (organic) phase (vortex)

                Spin 2 min at 12,000 x g (may go longer)

                Remove supernatant

                Air dry pellet (can be stored at -20 C). Can be speed vacuumed to dryness.

Note that all “X volumes” are the volume of the original protein sample. So if you have 100 ul of sample, in step 1 above you would add 400 ul MeOH vortex, then add 100 to 200 ul Chloroform and vortex. Then in step 2 you would add 300 ul dWater… and so on.

As for the silver staining issues, if you are having high background it often indicates that your dishes are not clean and rinsed enough. Silver staining is very sensitive and will pick up trace contaminates (especially detergents) very easily. You must have very clean containers that are thoroughly rinsed clean. Not sure if you are pouring your own SDS-PAGE gels, but if you are you have to thoroughly clean and rinse the glass plates as well since they are going to be in contact with the gel matrix.

Dear sir

Thank you so much for the suggestion.We will try as per your protocol

Dear Dr. Carroll

Our 2D image came out well as per your suggested protocol.

Thank You so much.

With the methanol-chloroform precipitation protocol provided by you, it would be never possible for us to obtain a good 2D image.

Our gel image is attached. please give your suggestions on the image.

Regards

Dr. Divya Jose