12 replies to this topic

[science noob]

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?

[BioMiha]

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.

[science noob]

BioMiha, on 29 November 2011 - 10:37 AM, said:

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.

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.

[protolder]

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

[bob1]

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...

Edited by bob1, 05 December 2011 - 07:11 PM.

[science noob]

bob1, on 05 December 2011 - 07:11 PM, said:

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...

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?)?

[bob1]

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.

[science noob]

bob1, on 08 December 2011 - 06:42 PM, said:

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.

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?

[bob1]

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.

[science noob]

bob1, on 09 December 2011 - 03:29 PM, said:

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.

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.

[Biouday]

Western blot is Semi quantitative because you are not measuring exact number amount of expression level of your protein.

But you can express expression in relative. To do run equal amount of protein and do western blot of your protein of interest and actin or any hose keeping protein and then measure band intensity using Imagej ( a free software you can use it measure band intensity) Then finally you can  express your result that your protein of interest increase or decreased relative to the house keeping protein which you checked

Edited by Biouday, 11 December 2011 - 03:45 PM.

[science noob]

Biouday, on 11 December 2011 - 03:44 PM, said:

Western blot is Semi quantitative because you are not measuring exact number amount of expression level of your protein.

But you can express expression in relative. To do run equal amount of protein and do western blot of your protein of interest and actin or any hose keeping protein and then measure band intensity using Imagej ( a free software you can use it measure band intensity) Then finally you can  express your result that your protein of interest increase or decreased relative to the house keeping protein which you checked

That was what I have been doing in terms of 'quantification'.  It is more of a relative 'quantification' more than anything else.  For this, we have to make an assumption that the housekeeping protein is equally expressed in all cells.

[Biouday]

if your protein of interest not interfering with housekeeping protein then the results are valid. To make sure you can check two or three housekeeping protein to get valid conclusion