If you do not have a pure protein of interest, you cannot obtain its "accurate" concentration.

It is not clear if you wanted "six techniques to study protein changes" or "changes to six proteins". If it is the former, there are many techniques that address many properties of proteins; some require pure preps while others don't. Over aging, many changes can occur to a protein: the level of expression, post-translational modifications (these can be simply numerous), folding, proteolysis etc. If you know what exactly you are looking for, it will be easier for you to find a way.

Thanks, Anil. 

 I want to find out change in concentration of six different proteins due aging in muscle tissue. I am performing western blot right now for all this proteins and I have specific primary and secondary antibodies for all of them. Is there another technique available to get accurate protein concentration? I don't have purified proteins to use as standers so can't perform ELISA. 

Are the proteins available individually or as a mixture?

You just want to see the concentrations? nothing else?

Hi Preeti, 

Try going for  MRM (Multiple Reaction Monitoring) for your 6 proteins of interest. It's an MS based technique. That would be quite beneficial, it would give you both absolute as well as, therefore, relative between two conditions., viz., aging vs young.

You could try cytometric bead array assays (CBA) that actually can let you determine the concentration of all six proteins at once. Ofcourse you need a flow cytometer for that. Just check to see if BD Biosciences has an array for the proteins of your interest.

You could use a Luminex type bead based assay if commercially available. These can be customized for proteins of interest.

Hi,

I don't see the reason to have absolute concentrations, hence Western blot or Mass spec are all only relative and to know the concentration in µg/gramm tissue would not improve the result. So you could use Elisa in case you have good positve and negative controls and you woud have to use a normaliser and always put all sample to be commpared onn one plate. Then t should work

regards

Sven

Have you tried to determine expression rather than protein concentration? The qPCR would help as it is straighforward and you could even determine a relative (or quantitative) expression of the mRNAs for the six proteins... 

Other option is use ELISA and express results as relative concentration but it would require a determination with a normaliser (e.g. Actin) as well pointed out by Sven...

Preeti,

      You might look into the possibility of using agarose gel electrophoresis .  It would seem to me that age induced concentration changes would be measurable through rates of protein migration.  I hope this helps.  Bob Corrigan

There is no reason to get accurate concentration of your six proteins in your experimental samples--at least as far as we understand from your description. All you need to do is simple and straight-forward: WB for your proteins and quantitate them "relative" to a known, supposedly unchanged, reference, such as beta-actin or GAPDH or histone H3. There are several band-quantitation softwares available for free (for instance, ImageJ). If you simply want to know the quantitative changes of the proteins over aging, I do not see why you want to use other fancy techniques. Common/basic lab practice would be enough; such as a careful protein estimation of your samples, loading equal protein amount, and consistent handling/processing of each tissue sample for lysis/extraction. If your goal is bigger than this simple notion that we get from your description, then feel free to detail your concerns and someone will come forward to help you. The more elaborate you are in describing your problem, the greater is the likelihood of getting the correct suggestion.

Western blotting is, in best cases, semi-quantative, irrespective of the quantification software... (no matter if it is commercial or open source). We experienced a lot of cases in which western blotting yielded nö result where HPLC, MS or ELISA Kits prooved to be more sensitive in a much higher resolution)..We tend to usw Western Blotting just for qualitative questions rather than quantitative ones....

Interesting and perhaps obligatory isoleation (hydrolysis of the tissue)  amino acids and exact analysis. Wishes good successe Zet. Dabrowski

I was trying to mix my sample to use them as standard for all the gels. I have homogenates of tissues with different concentration. I don't know how to mix them together to get same concentration of each in final volume. Can you please suggest me how to achieve this?? 

Preeti, I had said earlier: "The more elaborate you are in describing your problem, the greater is the likelihood of getting the correct suggestion." That still holds true, even now.

Now you say, "I was trying to mix my sample to use them as standard for all the gels." Why would one do so? How does "mixing samples" become "standard" for gels? What exactly do you want to do here?

Then you write, " I have homogenates of tissues with different concentration. I don't know how to mix them together to get same concentration of each in final volume." Are these homogenates from different tissues representing different samples? If yes, why mix them together? If they are homogenates from the same tissue representing the same sample (but in different portions--say, for example, the tissue was too large to fit in one tube and you processed it in parts), then why mix them together? I cannot imagine an experiment, involving an SDS-PAGE later, where multiple samples are "mixed together" in equal proportions (though for special experiments like SILAC there is a need to mix two samples in equal proportions, but that is irrelevant here)

It is clear that you are facing a problem. But I cannot understand what it is, unless you find a way to convey your problem clearly. Maybe someone smarter than me will come forward to help you.

What I guess I understand is this: you have extracts of varying protein concentrations (fairly natural) and you want gel-loadable dilutions where all your samples are of equal concentrations--so that you don't have to reset pipette settings for each sample. And you want to know how to do that. Is it so? Or, is it more complicated than that?

by amino acids analizer

You should read an excellent new paper about important factors for quantitative immunoblot analysis. It recommends use of multiple internal loading controls, for maximum precision and reproducibility. Also suggests diagnostic experiments you can run to look for sources of variability.

http://stke.sciencemag.org/content/8/371/rs2.abstract

Thanks Amy!! very good paper. Really appreciate your help.