Never,

Fixing is essential before staining because in this step, many interfering substances such as IPG buffer and component of Rehdration buffer were removed. This substances gives poor sensitive gels or background upon staining. If you are not getting spot, you can stain the strip for confirmation of presence of protein before running of 2nd dimension. What kind of staining you are using, if Silver stain then your reagent purity can matter. For silver staining, it is necessary to use high purity reagents with MQ water (purified morethan 15 ohm).

The problem could be the second dimension which is due to the electrophoresis tank leakage! You may want to check that too!

If you are fixing the gel after the first dimension then yes this would be the problem - fixing is essentially designed to immobilize the proteins to prevent any additional diffusion of the bands - immobilizing the proteins means they will not migrate in the 2nd dimension - do not fix the gel between dimensions.

If you stained with silver, you may de-stain the gels and re-stain it again. Some spots may come out, but this may give you a higher background. So, you have to monitor the developing step carefully. Worth to try. Also, as mentioned by Narendra, high purity of water is necessary (~18 ohm). Besides staining steps, please also make sure there is no gap between your IPG strip and the 2-D gel during electrophoresis.

It is a bit hard to answer without a more detailed description what you have tried already and how your experiment looks. What material are you loading? what is the protein concentration? What do you stain with and how do you visualize?

In my opinion, fixing a gel is only nescessary if you want to store it for a few days. If you process the gel immediately, no fixing is required at all.

I would start with checking if your 1st dimension went well by staining your IPG strip with Coomassie. You should see clear protein bands on your strip. Next, check if the transfer from 1st to 2nd dimension worked by again staining your IPG-strip after transfer to the 2nd dimension (the bands should now disappear).

Last, make sure you actualy have enough protein in your sample to visualize. If you are using serum, make sure you are loading about 1mg/mL on your gel. If you have (semi-)purified samples, this can be a lot less, but you will need at least 10-50 ng protein in your spots to be able to see it at all.

If fixing after first dimension is not made and run second dimension, there is all possibility of getting spots on 2D gel.

If you are not getting the spots even if you are not fixing, then the proteins that you are separating must be highly basic proteins.

Check that!!

did you measure protein in your samples. some time low concentration of proteins produce faint of non visible spots within the gel but appear during immunoblotting. so please be sure your samples have the sufficient amount of protein firstly and then try to run 1D gel and stain it with CCB before running your 2D

Hi Jimmy,

First check the expiry of your IPG strip. Next, if you are doing coomassie staining then fixing of gel can not be a problem (as dye itself made in fixing solution), But if you are doing silver staining then first put your gel for fixation for few hours and then proceed.

Another important step, is to check proper re hydration and absorbance of your protein occurred prior to iso-electro focusing. As, if IEF done properly then you should get spots on the 2D gel.

Do silver staining first, as it is more sensitive and can stain gel containing very less amount of protein, this to check the pattern of spot separation. As, sensitivity of your coomassie statin and amount of protein matters in coomassie stating.

If u did not find any spots on after 2nd dimension..it should be due to anyone of the following...

1) Very low concentration of protein in applied sample

2) No absorbance of protein at rehydration step (check if u are keeping the gel side of strip towards buffer)

3) The protein on IPG strip not transferred to gel during faulty 2nd dimension run (check if u by mistake placed cathod pin into anode slot on powerpack)

4) Not working of ur either staining or developing reagents