Is western quantitative? - (Nov/05/2007 )

I am trying to demonstrate different levels of expression of my protein of interest in a set of different murine cell lines. I am using actin as a loading control. In order to have some numbers, I am dividing the densitometry value of my protein by the actin one. Is this the right way to do it? Is western blotting in fact a reliable quantitative method?
I would appreciate any comment about this. I want to get the most information possible from my images.

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immunostaining of house-keeping proteins is widely excepted to demonstrate equal loading of protein amount per slot; but why are there differences in loading?

to compare it to other specific immunostains, you can take it only for the linear range of each immunosignal; this linear phase should be determined by calibration

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It's not quantitative, at least that's what I was told. You can show the protein levels by showing the blot, and people see it's more or less, or not present, but it's not really quantitative. Too many variables, even in film developing, especially if you do it in cuvettes, not in the machine. Well, maybe if you do the antibody calibration experiment to know when your signal is in the linear range, as the Bearer said, but I wouldn't go for it. It's a drag. In general, you don't go for numbers, just fine looking bands.
Why don't you do RT pcr instead?

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hi,
WB is absolutely semiquantitative, there is no doubt about it.
of course always u can convince yourself saying that u have loading controls (in ur case actin). But as other forum members mentioned the intensity of signal depends on several factors begining from transfer,Ab conc and developing. But developing process is a crucial one, if you want to compare different experiments u need to expose the film such a way you get same intensity of signal for both your protein of intrest and loading control.
if u ignore this fact then you may get highly variable results, which may be at the end insignificant, but the relative difference will be the same.

in summary it is very difficult to get a quntitative data unlike ELISA where you have standard curve.

welcome for you comments

sravan.

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Western blot can be used as a quantitative method. But you have to use the correct readout. Scanning a film and looking at the bands with programs like photoshop surely isn´t the best way. But if you have a chemiluminescence camera or something similar, which actually counts photons, than you "just" need a standard. This can either be an internal control as actin or an external like the purified protein of interest in a known concentration (for absolute quantification). As you want to compare expression levels in different cell lines/tissues, I would go for the second (external) control, as also actin can vary from tissue to tissue in its expression level and thereby also a ratiometric determination would not help. Alternatively you could add another purified protein which is not present in the sample itself as an internal control to each sample. But be aware that you load equal amounts of total protein for the western!

All of this is only quanitative for getting ratios! Getting real numbers like µmol etc. you would need methods like ELISA. But I guess you meant ratios when you were talking about quantifying your protein.

For quantifying expression levels via RT-PCR I have mixed feelings even though a lot of people are using it.
BUT: with RT-PCR you only quantify mRNA levels and not expression levels of the protein. And it is reported quite some times that mRNA levels not necessarily correspond to protein levels. Even an upregulation of mRNA does not always result in an upregulation of protein.

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Complete rubbish,

Quantatation can only be measured in SI UNITS....what is the accepted SI UNIT for densitometry?

I know what it is for distance...metres, centimetres, millimetres etc
I know what it is for weight....Kilogrammes, grammes, milligrammes etc.

WHAT IS THE SI UNIT FOR DENSITOMETRY ?

The QUALITATIVE estimation of bands in western blotting is QUALITATIVE. Actin or tubulin are used to approximate equal loading.

Please prove me wrong?

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I agreed.

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You may wish to do flow cytometry.

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hi,
HOW CAN IT BE COMPLETE RUBBISH???

follow me carefully.....
you take known amounts of protein of interest (3 different conc).
perform western blot with unknwons and knowns (above 3 diff conc).
after developing the film analyze the signal internsity with densitometry.
assign different densities (numbers) to the known concentration of protein (like standards in ELISA) then
collect the densitometry values from unknown samples similarly and correlate them with the standard densitometry values
convert those unknown denistometry values in to ng or ug or mg with respect to the above 3 different known concentration(stds).

hi this is what exactly happens in the ELISA. you convert the end point color intensities which does not have SI units into the values which have SI units with the help of standard.

at the end if you use standard in the western blot (OFCOURSE WHICH IS A TEDIOUS PROCESS) you can quantitate the results.
ofcouse i would not try to do this way, instead i would go for ELISA.


open for comments
sravan payeli




Complete rubbish,

Quantatation can only be measured in SI UNITS....what is the accepted SI UNIT for densitometry?

I know what it is for distance...metres, centimetres, millimetres etc
I know what it is for weight....Kilogrammes, grammes, milligrammes etc.

WHAT IS THE SI UNIT FOR DENSITOMETRY ?

The QUALITATIVE estimation of bands in western blotting is QUALITATIVE. Actin or tubulin are used to approximate equal loading.

Please prove me wrong?
[/quote]

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Dear friend

U can use W.B as a quantative tool only to present differences, its not accurate.

The important thing when u use densitometry, its better that the results would be obtained from the same film developing

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