I would consider proteomics analysis. It needs lots of work though on mass spec. Another thought, but would need lots of courage lol. Using Flow cytometry, you can have the labelled antibodies for 5 proteins at a time with 5 different colors, the more colors you can have the better for you, and then you run them on HTS and it would take a week or two after optimization to get things done. The tricky part would be labeling the antibodies with flurescent tags if you cant purchase them tagged already. Five colours for five proteins, you will need to run these samples 4 times, and these four times can be just optimized on a small scale before running the whole thing.

Now the question that remains, how much are you willing to spend? 

Hope it is helpful.

Cheers

A

I would consider proteomics analysis. It needs lots of work though on mass spec. Another thought, but would need lots of courage lol. Using Flow cytometry, you can have the labelled antibodies for 5 proteins at a time with 5 different colors, the more colors you can have the better for you, and then you run them on HTS and it would take a week or two after optimization to get things done. The tricky part would be labeling the antibodies with flurescent tags if you cant purchase them tagged already. Five colours for five proteins, you will need to run these samples 4 times, and these four times can be just optimized on a small scale before running the whole thing.

Now the question that remains, how much are you willing to spend? 

Hope it is helpful.

Cheers

A

Search for a biostatistician expert in protein analyses and pick up the best 5-10 significant proteins for validation by Western blots and pathway analyses.

If you can switch to ELISAs instead of Western blots, you can increase the throughput by using 384-well plates, which have enough wells for all the samples in duplicate (360 wells) + standards (24 wells). You would then need one plate per primary antibody, 20 plates in all.

As Anwar said, The best method is proteomic in which whole of proteins are evaluated. One of the best technique is 2-dimensional electrophoresis (2-DE) that can be performed easily. Albeit, this technique need some expensive instruments and also needs further confirmation techniques.

However, you cal also use DIGE, if the related instruments are available in your Lab.

If you need any help or have any question about 2-DE, please dont hesitate to contact me.

Br

Hi Nicole

The question is a bit vague and you don't give enough information to really know what you are up to.

Changes in protein levels can be very subtle or could be large changes in the order of 10 - 50 fold increases or even decreases when biological systems are challenged or altered. These changes can be detected specifically by using antibodies raised against these proteins as you intend doing by western blot but subtle changes are not easy to quantitate. Using ELISA as suggested by Adam is possible because you have the antibody (western blot uses antibody against the same proteins) but you may need to bind your antibodies to the plates yourself and have to validate the success of these assays. ELISA is however probably the best way to detect small changes in protein concentration.

You can also go the mass spectrometer based protein quantitation using tagged or label-free techniques where you could relate the specific proteins to a common background or housekeeping protein to get relative changes in levels in the sample. The problem is that you could probably do all 20 and even more proteins per sample but small changes in the levels are difficult to quantitate and less than fold changes are difficult to quantitate but relative quantitation could provide the information you are looking for.

Both techniques require accurate work and have many points where things can go horribly wrong. They are also expensive, so you should make really sure about the proteins that you want to assess and see if you cannot get pointers of pathways that are being altered and look for the critical proteins in these pathways which could reduce the number or proteins being assayed.

Planning will be very important no matter which route you go.

Hope this helps a bit.

Good luck

Duncan

You may consider suspension bead array method by flow cytometry. this method is popular for multiplexed detection of cytokines and chemokines from culture supernatants or body fluids.

By this method you can look for change in the level of multiple proteins simultaneously from the same sample and 20 is not a big number for this. Further the whole daily sample preparation and data acquisition for 60 samples would take 6hrs . However it has got bottlenecks

Expertise in in the custom bead preparation if such beads are not available commercially 

You will require two different antibodies per protein (preferably monoclonal)

In case you find this method of certain interest then I can elaborate further.

If the samples are serum/plasma, then taking the Luminex multiplex approach is the best option. there are now solid phase arrays for several serum proteins. 

http://www.hindawi.com/journals/bmri/aa/326918/ref/

The FM3D reader from LUminex caan handle96-well and 384 well plates easily. test the samples to be in the linear range of the standard curve and you are good to go. the testing may lead to you breakdown the panel into sub panels based on the dilution of your samples to fit into the linear range of the standard curve. 

If you have the 20 antibodies, you can try an approach equivalent to slot blot. This is an easy procedure that will give you a general idea about the expression of the different proteins in your samples. Further, you can perform SDS-WB analyses. 

Hope this will be useful

Hi Nicole,

i agree with most of what was said before. WB is just not very suited to high throughput approaches. That said, assuming that you HAVE to use SDS-PAGE/WB, you can use something like this (http://www.licor.com/bio/products/accessories/mpx/). We have used this in the past to do something similar, although not on your scale. It requires a lot of sample, so if your sample is a limiting factor this is not an option. The MPX system does 24 AB lanes on a single mini gel membrane that was loaded with a single sample, combined with two color detection if you  have the scanner.The problem here is that comparison between samples is very problematic. Blotting can be uneven on a single membrane, and comparing 60 individual membranes will most likely be impossible. Maybe if you can prelabel you samples and create an internal standard that is identical over all lanes and membranes? Similar to the normalization approach for 2D-DIGE? Overall i'd say this is not going to work using conventional WB.

One other alternative would be to spot our antibodies onto a substrate (glass slide) and use them as an AB array. This approach can support higher throughput. 

Good luck!

Hi Nicole,

any antibody-based methods will depend not only on the availability of antibodies, but also on their quality (specificity / sensitivity). Depending on the method that you are using, you will also have problems with the dynamic range of your assay (relevant, e.g. for the Western Blot, possibly less so for the arrays that Christoph proposes. You do not say, what kind of proteins you are looking at - solubilising them in a buffer that is suitable for Ab capture arrays might be an issue.

I would strongly argues for the suitability of proteomics shotgun methods for your problem. There are now a variety of solutions that have been published in the last 10 years or so that you could adapt to your problem. The keyword that you are after is "targeted quantitative proteomics", some of the techniques that would allow you to do this are SWATH, MS^e (MS to the power of e), SRM (in the old days this was called MRM and a lot of oldies still refer to the technique as that). One particular useful (open source) software package that could help you with setting up and interpreting experiments with all these techniques is Skyline from Mike McCoss's group.

https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view

I have the same opinion, Proteomics probably is the best way to analyse protein changes. It´s necessary a lot of hard work and patience and very careful to not contaminat your samples.

An additional thought on the mass spectrometry-based proteomics option: You should probably only consider the MRM (multiple reaction monitoring) option with 180 samples to analyze. The cost for the isotopically labeled versions of the targeted peptides (required as internal standards for quantitation) will not be too bad spread out over a relatively large project, and you'll be able to use cheaper-per-run triple quadrupole mass spectrometry instrumentation in your mass spectrometry core facility. In contrast, the shotgun route will require expensive isotopically labeled reagents and high-end mass spectrometry instrumentation. Furthermore, results will be relative rather than absolute concentrations and changes in the order of 20% will be too small to reliably measure (changes under 20% can be measured, but not usually reliably with real samples).

I think the suggestion of Christoph (Antibody microarray) is very good, but probably it will cost a bit... Therefore you will have a better quantification than with western blot.