In our lab, we have the Odyssey System and it works pretty well. It's less sensitive than doing ECL blots, but works really nicely for quantification, especially with good antibodies or nicely expressed proteins. I don't know your alternative systems, but with the Odyssey you can also scan other things like FLISA 96-Well plates (ELISAs detected with fluorescent antibodies). And the software is quite easy to handle.

Best,

Cindy

We use odyssey and simple westerns both alternating. I feel more than the system, good antibody does the trick of reproducible quantification.

Hi Michael, we use the old Amersham system, the LAS 4000 Mini, which was actually invented and built by Fuji, and it's a very robust and reliable system. Now it has been replaced by the Amersham Imager 600, which comes in the basic version for ECL only and a probably much more expensive version (the 600RGB) that allows also fluorescence imaging, that for accurate quantification purposes is probably more indicated, although you will struggle with the quality of the antibodies much more (as already pointed out).

I'm not familiar with the Licor system, although we screened many systems before buying the LAS and none looked as straightforward and simple to use...

Simple Western is a new way of running a western blot, which avoids the protein transfer.... I'm not familiar with it, but in principle it is quite interesting, since it should reduce variability, although I'm sure once one starts to work with it the flaws will come out and the specs don't actually seem to give a highly improved reproducibility compared to standard WB techniques...

Odyssey with the capability for both infrared fluorescence and chemi would probably be your best bet.  Odyssey blots are cheaper vs chemi blots but it would be nice to have to the option to do chemi when femto sensitivity is needed in your lab.  In my experience Odyssey infrared fluorescence has pico sensitivity but it is possible that I did not have my secondary concentration high enough.  I typically use 1:50,000 licor secondary dilution for most of my licor blots.  Sometimes I use 1:20,000 for lower abundance targets.

To Brian's point, I use 1:15,000 for LiCor secondaries and my target is still sometimes difficult to find compared with a parallel assay done with ECL. LiCor is great for multiplexing when true quantitative analysis is needed, but the background can be a hindrance. ECL is super clean when compared with the issues associated (and too often experienced) with IR autofluorescence.

You can try Cleaver Scientific. Their western blot system is similar to Bio-Rad and works just the same, at a cheaper price. 

I have switched from using ECL to Odyssey, since it feels much more reproducible (of course, it depends on samples). I like imaging dried membranes instead of wet, since it improves noise-to-signal ratio by several fold (and also this way I can prepare multiple membranes, dry then, and image in few days  without any problems). My husband also uses Odyssey to image Zebra fish and the fluorescence within their gut. The images look amazing :) So I think using Odyssey has lower sensitivity when it comes to common western blot samples, but it has more imaging options. So you have to think carefully about the applications you need the system for. 

I would like to clarify that the Amersham WB system is more than just an imager. It is an integrated system for electrophoresis, gel scanning, transfer, probing, membrane scanning and with built in evaluation software. It is based on fluorescent detection and multiplexing (Cy5 and Cy3) that is the best option for quantitative results that was already mentioned above. The whole workflow in standardized and focused on quantitation and reproducibility. The samples  can be pre-labeled with Cy5 and the Cy5 total protein protein signal used for normalization. 

Science Journal discuss this subject: Western blotting a guide to current method from June 2015. Different methods to obtain quantitative results are discussed as well as common pitfalls. See link

http://science.imirus.com/Mpowered/book/vscim15/i4/p1

I am afraid that apples and oranges are compared. Amersham 600 is an excellent system as well as older LAS 4000 ( in fact better), but these are imagers, just as Licor is, in fact. The only system that really covers tasks required to have truly quantitative WB is Amersham WB among instruments mentione. Read carefully what Amersham WB is and how it works to understand that your job is to feed it and the rest machine will do to make sure that gels are the same, conditions of electrophoresis, chemistry etc. so that REPRODUCIBILITY in electrophoresis, transfer and blotting is absolutely ensured. Even labeling, as pointed with Asa, is done in a way to minimize matrix effects, ensure wide range of signals, reduce interference etc. with cycanine labels used in 2D or sequencing. The essence of quantitative WB is in absolute reproducibility of all steps needed to make final stained membrane. Imaging is just a part of this. All these provided that you really need true quantitative WB. Sensitivity is also more a marketing thing than a real value. What is really important is LINEARITY of optoelectronic response. These response curves are never linear and they specially tend to curve at the bottom (low signal) and at the saturation end (very high signal). Cyanine dyes give extremely wide OD range and that is why they are used, to correspond to wide concentration ranges in gels/blots. I would suggest really careful preparation in decision making process what to order. 

We would like to add two things to the discussion:

a) using fluorescence (e.g. red/IR fluorescent secondaries) is wonderful and should be the first choice. However, the sensitivity of fluorescent antibody conjugates is not sufficient for low abundant target (if somebody know a solution to this, go sell it for a fortune). Chemiluminescence is a chemical reaction with all its disadvantages however it is (except radioactive labeling) the only possibility to detect low abundant target expression. To have all options, we think that the ideal imager should be capable of detection both, red/IR fluorescence and chemiluminescence with great sensitivity.

b) reliable quantitative Western Blot Analysis is still an issue and all recent papers show, that normalization of the target protein expression using so called house keeping proteins does not work. From our point of view is the only solution, that you normalize your target protein with co-detected total protein. However, the total protein must be detectable with high sensitivity at the time, the target is detected. When the total protein is visualized using fluorescent labels, samples must be cleaned up first (time consuming, expensive) or labeling efficiency must be monitored. The Smart Protein Layers technology uses a combination of fluorescent internal + external standards and fluorescent total protein visualization.

You may find out more at http://www.dyeagnostics.com/site/en/products/spl/

Keywords: quantitative Western Blot, reproducibility, Western Blot, IR fluorescence, chemiluminescence, normalization, reliability

We are using in our lab the ProteinSimple system, WES.

The thing is that, although it is called Simple Western in my opinion is not a fortunate name. It is a capillary electrophoresis, with the subsequent detection by immunoassay and chemiluminescence. At the beginning it took us a bit to change our mind, but now we are really satisfied with the results.

Main advantages:

- low volume of the sample required- you need only 4ul of the sample, and depending on the expression of your protein this needs to be more or less concentrated.

- At the end, although it sounds scary to use antibody dilution like 1:50 without the option of re-utilizing Ab, as many labs do, we spend much less on antibodies. Normally, if possible, we buy only a sample size, since you need only about 8ul of diluted antibody per well.

- As to the quantification, it takes some effort to have it optimized for each of the proteins- the linearity, LOD, LOQ. But the automatization of the whole process makes it easier. You don’t have biases at the gel resolving, transfer, Ab incubation, detection, etc. The only parts that a human error should be considered are the preparation of the samples and the antibody dilution. From the analytical point of view we find the method reproducible, and the sensitivity depends on the target protein.

By the way, the biological normalization is always a problem and depends on the model of the study. You can use a protein X and other person that uses Y will claim you are wrong.  You can also use the technical normalization of the system, if you need.

- You are quantifying peaks and area under the peak and not the density of the band. The band- view is only a method to picture your results. You can see the level of the background and the points that are used to calculate the background- if you get more advanced and comfortable with the program you can play with this values as well.

- The training of the technical personnel is not complicated.

- Everything you need to do the run is supplied in the kit, you only need to have your sample and Ab.

- The run takes really only about 3.5 h. So, in one day you can analyze many samples- in one run you analyze 24 samples. The sample preparation, once you take a rhythm is not more than half an hour.

- You can quantify total protein, if you like, in the same system- but within a separate run.

Now, disadvantages:

-          It’s not a WB- the matrix is distinct, the volumes are distinct, the buffers, etc. So, some proteins might migrate different than you are used to in WB.

-          The analysis of the results, if you need them to be truly quantitative, is quick BUT requires training.

-          The cost of the consumables.

You can take a look at our poster if you like,

http://www.proteinsimple.com/literature_download.html?docid=1424

Hello Michael! In our laboratory we use Wes by Protein Simple. At the beginning we used WES in parallel with classic WB to integrate and confirm the results and it worked well. It is convenient since you will have to use just 5 μl of your samples to obtain very precise quantitative data. Moreover the antibody database is increasing each day, so now it is still easier to work with it (some of classic WB Ab is compatible with WES technology). The whole process lasts for just 3 hours, so it will make you save a lot of time. What we find also very useful is that results will be available not just as the classical band, but also as a peak of the chemiluminescent signal, which is pretty interesting to give a final complete evaluation in order to obtain a quantitative analysis. In the end, the samples, reagents and Ab preparation is very simple and quick (it depends by number of samples). I compare the procedure with the real time PCR where you have to prepare your reagents (primers, TAq, etc...) and samples and put your plate into the instrument, it is going to take care of everything!