What is your staining method? If you use Coomassie try silver staining.

Which precautions do you take against protease activity? What is the composition of your IEF buffer?

Answering these questions could help with finding a solution for your problem.

Cheers, Christian

What is your staining method? If you use Coomassie try silver staining.

Which precautions do you take against protease activity? What is the composition of your IEF buffer?

Answering these questions could help with finding a solution for your problem.

Cheers, Christian

 Hi Leonardo.

as Christian says, it appears that you are using Coomassie for staining your gels, so you are just looking at the tip of the iceberg, due to the low sensitivity of this stain: in practice, you can only see the most abundant proteins in your sample. In order to increase the sensitivity, you should change the detection method (silver stain in much more sensitive) and might allow you to recover low-abundant proteins. You should use a protease inhibitor cocktail to avoid protein degradation, even though looking at the gel the spots are nice and there is nothing at the low-MW end of the gel.

Cheers,

Alberto

Christian, Thank you for responding so quickly.

I used Coomassie.

I did not use any protease inhibitor.

The composition of my IEF solution follows:

Urea 7M

Thiourea 2M

CHAPS 2% (w / v)

Bromophenol 0,002%

IPG buffer and DTT added at time of use.

Hi Alberto

agree with you in relation to sensitive with silver staining, but as far as I know, the silver staining is interference by the time I need to sequence the samples by mass spectrometry.

Hi Leonardo,

you could use the cOmplete Protease Inhibitor Cocktail from Roche

http://lifescience.roche.com/shop/products/complete-mini-edta-free-3271382-1

but I don't think that it is absolutely necessary.

Your IEF buffer looks good to me and the thiourea will also inhibit protease activity.There are silver staining protocols that are compatible with mass spec analysis (please see the attached publication).

Cheers, Christian

Article A mass spectrometry compatible silver staining method for pr...

Hi Christian and Alberto, I also did a gel with the same treatment using 1000 ug of proteins to see if I could view more spots. Sending the gel attached.

Hi Christian, thanks for the submitted paper and the link, I read today. It has a very interesting paper also he uses aldoses in place of formaldehyde, to reduce the interference when sequencing samples by mass spectrometry.

Cheers, Leonardo

http://onlinelibrary.wiley.com/doi/10.1002/pmic.200800321/abstract

Hi, 

maybe you can load more protein to IPG strips. Because conventional protein concentration measurement methods gives us only total protein concentration. So abundant proteins cover the minor ones.  Consequently it causes only a few abundant protein spots on your gel. For 17 cm strips you can load up to 1500 ug crude extract. Changing the staining methos is not necessary i think. 

hope it will help 

Hi Leonardo;

In addition to the above comments, I would like to note that you have vertical streaking in the basic region of the gel. You would need to improve your sample preparation. It is the critical step in 2DE. 

Try to use the destreak reagent (GE healthcare) in order to improve resolution in the basic región. 

Do you have conductivity problems during run? Did the IPGphor arrive at 8000V during the run?

God luck

Use protease inhibitor and can dye their gels with Sypro Ruby

Thank you all for your help. Hi Nil Guzel, can be a solution to my problem. But this week, staining with a silver staining compatible with mass spectrometry to see the profile of low abundance proteins.

Hello Mario, I also realized the problem with horizontal streaks . In the near gel, will apply Destreak reagent in the sample to see if it gets better. With respect the run, look at the attached to all normal.