First of all see if the pore size of the gel is according to the protein size

Second are you running sufficient amount of protein on the gel?

Oxidation can often make large aggregates which have trouble entering ND gels, maybe a reducing ND gel would help?  You can see protein in stained (or blotted) stacker right?  

to expand on the above comments - what is the pI and Mr of your protein ? Also what is the pH of the PAGE system you are using

Thanks for your reply,i am working on 4 proteins,the pI of my all proteins is in between 5.0 to 5.5 and net charge at PH 7.0 is -9 to -22,three of them acidic(charge negative) and 1 basic(charge positive)molecular weight 80 to 35 kDa with GST tagged.The PH of my page is 8.8.Thanks