By using the proper controls (loading similar protein loads, using a reference protein, etc), you can tell over-expression, upregulation, downregulation, etc. To compare between cell types, that's where you need those controls. Load can similar amounts of lysate on each gel, and show that there are similar amounts of a reference gene in each sample. Once that's established, look at the signal from the target. Is there a difference in intensity of the target with similar intensities of the reference band? If so, there's a difference in expression. If target signal is really strong in both samples, dilute them down to see if one signal persists further as the dilution increases. The other option is to do quantitative PCR to look at expression levels.

Hi Eric,

Are your results reproducable? If so, then your extraction procedures have given you the answers already in the sence one does better than the other.

Good luck,

Ali

How do you know the samples have the same amount of protein? Most protein assays are terribly sensitive to additives, especially detergents. What are your extraction conditions? Usually integrated intensities over the whole lane are used to gauge protein amount and the band of interest is expressed as a proportion of the whole lane intensity. Some proteins absorb gel stain at very different amounts and appear darker or lighter so a purified standard quantified by some other method (UV extinction, AA analysis or dye assay) is often needed.

Dear Eric, after reviewing your queries, I notice that the problem arises in your extraction protocol so follow anyone sensible and reliable method for better extraction and then stain with silver staining. If again the problem arises, then go for purification process.

Second, most of the journal accepts qualitative gel image for publication but you must analyze the intensities of the gel band using available software (imageJ/ image studio lite etc.,)  and then plot a graph for that band.

I think Edwards is probably correct: there is interference in the protein assay from component/s in the extraction buffers. You don't say how you determined protein levels, but the blank for the determination must be the extraction buffer in all cases.

Edward and Geoff are right. Problem may lies in protein assay? You may use Ponceau S solution (before immunolabelling), Amido black (after immunolabelling) or any other stain and use entire lane to normalize your protein of interest.

it entirely depends upon goal of your specific step/study.

whether you are targeting whole cell protein or just a specific one

if you are aiming to compare extraction efficiency of various protocols for total protein, then Bradford/lowry assays are comparatively more accurate, economical and freehand methods for protein quantification..

if you are aiming to compare extraction efficiency of various protocols for specific protein, then you may goto purification way followed by protein assays(functional enzyme assays/immuno-assays.. again depends upon nature of target protein)