It seems to me as overloaded with pelleted proteins, and the sample buffer if insufficient, leads to incomplete denaturation and loss of resolution. Middle lane is just the remaining liquid after the protein pelleted.  

Degraded is fragmentation of the protein into a wide range of peptide chains vs. denaturation which is unfolding of the secondary or tertiary structure. It could be degradation is part of the reason as well as buffer depletion and overloading.

Protein overload, excess salt, or excess non-ionic detergent in a sample can do this. As indicated by Mangal, there is probably too much protein in this sample so you have both exceeded the available amount of SDS, resulting in incomplete denaturation and a variable SDS:protein ratio and overloaded the lane. Try a serial dilution of the sample using 1X sample buffer as the diluent.

Thanks for helpful responses. Help me to understand 'degraded'. If you leave a protein prep out at room temperature, it gets denatured and loses its activity. How does it get degraded? Repeated freeze thaws? Without proteases, what's the mechanism for fragmentation? 

Collagen IV is 160 kDa. If it was fragmented there would be multiple bands of low molecular weight with fewer bands bigger than 150kDa. But in the gel, the entire lane was stained evenly.

denatured means partial or total loss of native conformation. For example, SDS and chaotropk (i.e., Urea) denatures proteins by altering, and ideally linearizing, their 2D and 3D structure with no loss in the total number of amino acids.

Degradation refers to breaking down of the protein into smaller peptide fragments. For example, trypsin degrades a protein by selectively cleaving alongside a basic amino acid. Freeze thaw can both denature and degrade a protein (loss of structure and breaking the protein into smaller peptides).

In addition to the mechanical damage freeze-thaw can do to a protein, it can also has drastic effects on the ionic strength and pH of the solution - resulting in denaturation and aggregation of the proteins. Including a small amount of non-ionic detergent in the buffer can help minimize this.

Thanks Bruce. It was suggested to me that the gel indicates the protein prep was degraded by repeated freeze thaws. The purified protein prep would not contain proteolytic enzymes. I am intrigued what is the mechanism by which freeze-thaws breaks a protein into peptides? 

Freeze thaw cannot cleave the peptides but lead to denaturation, opening up of the 3D structure, which is easily accessed by the proteases and hard to avoid if the vial is opened outside a laminar hood, not filter sterilized and kept at room temperature (allowing microbial growth). The bacterial spores are present all around the lab, grow and release proteases. The degradation is slowed down by keeping samples at -20 and -80 or by adding microbial growth inhibitors such as sodium azide (present in protein Ladders). The microbes grow on favourable conditions such as thawing in absence of growth inhibitors.   

Hi Mangal

Thanks for the plausible mechanism. Will keep in mind to freeze the protein prep that is exposed to room air. This explains why commercial protein standards (usually BSA) can be stored at room temperature (denatured) but contains sodium azide.

But still, I'm still wondering what I am seeing in the gel because the collagen IV is meant for cell culture so it's sterile and we open it only in the laminar flow cabinet. No proteases. Besides, proteases should cleave into small fragments, so I should see lots of small bands in the gel, but I don't. I see the entire lane lightly stained including at protein sizes bigger than expected. What can Coommassie stain aside from proteins?