Western blots are 'semi' quantitative? - (Nov/29/2011 )

What does the "SEMI" quantitative mean? that it doesnt fully quantify proteins?

With the softwares to analyse band intensities and volumes, would that be fully quantitative?

How common is it to use westerns to quantify protein expression?

It means you need a standard curve with known quantities to compare your unknowns to something. That is why it's semiquantitative. In quantitative methods you measure or quantify a quantity directly e.g. weighing or measuring time. No software can help you. It is what it is.
It's pretty common to use western to quantify protein expression as long as you are aware of the shortcomings.

BioMiha on Tue Nov 29 18:37:25 2011 said:

Would you always need a 'standard curve' for WB analysis? Or can you just use enough appropriate control - housekeeping protein ratio and internalised control? The software I use measures band volume/intensity.

Hola, the above answers are to know protein concentration against band intensity, and probably you´d better having an standard curve, but in other cases as rate of phosphorilation of proteins, that I imagine is your field, by other of your questions in the forum, here we compare intensity of phosphorilated protein band against dephosphorilated band and seeing by this way the rate of inhibition of any molecule, for more accurancy you could relate this to the actin band as control of loading but it isn´t necessary, to me, if the amount of loaded protein is similar

You can try, but you will find the results are very variable. You need to be able to load the same amount of total protein into each lane, transfer it all evenly, and then get nice exposures that are not over or under exposed consistently for each membrane that you run. I have found it to be next to impossible to get exactly the same (or even similar) results for the same lysates run at the same time on different gels, transferred at the same time in the same blot apparatus, and probed in the same solutions with all conditions identical...

bob1 on Tue Dec 6 03:11:06 2011 said:

What about using a house-keeping gene as control? E.g. actin, tubulin, GAPDH.

I was also told to run control lanes which are common to all membranes (e.g. use a positive control) to control any handling differences between gels.

My other question is: When running multiple transfer chambers, what is the settings to look out for (i.e. constant current or constant voltage?)?

Try it and see -my personal experience (10ish years) is that it doesn't work between gels, no matter how good you are at westerns, even with housekeepers, unless you can discount the variations in transfers, exposures, antibody steps, washes, etc.

bob1 on Fri Dec 9 02:42:38 2011 said:

Thanks for the advice, bob1! 10ish years doing western blots makes you a pro!

So, you wouldn't use westerns to quantify protein but just merely using it as a qualitative indicator if protein expression is up or downregulated based on band intensity? So, say if I have n=10, I would run n=1 as a "representative blot"? or run all n=10?

Yes, qualitative is what I would call it. It definitely works for general trends, but you won't get absolute concentrations out of it, there are just too many variables to work with. I suppose it might work if you could add known quanitites of a certain protein onto the gel and comparing, much as you would for qPCR.

bob1 on Fri Dec 9 23:29:15 2011 said:

Some thing like a standard curve with say BSA.

The only method to quantify protein expression that I can think of is ELISA. This will definitely be fully quantitative. Cell biologists are hesitant to use flow cytometry because the condition of the cells are somewhat different when analysed (in suspension). Immunostaining is qualitative as well (unless you have a good software to count stained vs unstained cells). However, if both cells are stained, don't think you can tell staining intensity (over/under expression) between them.