I am beginning a new research project and trying out Western Blotting for the first time. I plan to analyze the protein expression of EAAT-2, mGlur5, NMDA-NR2B, and a few other proteins intimately connected to the glutamate transport mechanism in the brain. Is it possible within a single western blot to visualize a band for each protein in the same column? I'm concerned that the application of particular antibodies may interfere with ones for other proteins. I assume here of course that all of my proteins have different weights, so the bands would not overlap.
I've seen papers that do simultaneous western blotting, but does it always work or is it dependent upon the particular combination of proteins and antibodies?
In addition, is RT-PCR (Quantitative, not Real-Time) limited by the same thing?
Jay: I would not recommend this if you are just starting out with Western Blotting. What you are asking can be done, but you really have to know your antibodies very well. Many antibodies will bind to other proteins (non-specific binding) and you could get into trouble knowing for sure if the band density for the protein of interest is due solely to your anitbody+protein of interest or to your antibody+protein of interest+non-specific binding.
I would recommend initially running WBs with each antibody separately so you can get a feel for the specific and non-specific binding characteristics. An alternative approach would be to cut your membranes so that each cut membrane only contains the molecular weight protein of interest and then do single Ab WBs on each. Another alternate approach would be to run one WB with one Ab, then strip the membrane and re-probe with your next Ab. Then repeat for the third. I've successfully used this approach many times with good results (although stripping the membrane will reduce some of your protein content and could affect the subsequent Ab binding; so you need to consider that). As I suggested above, you should run individual WBs for each Ab initially until you fully understand the properties of the Ab.
I hope that helps.
Jay: I would not recommend this if you are just starting out with Western Blotting. What you are asking can be done, but you really have to know your antibodies very well. Many antibodies will bind to other proteins (non-specific binding) and you could get into trouble knowing for sure if the band density for the protein of interest is due solely to your anitbody+protein of interest or to your antibody+protein of interest+non-specific binding.
I would recommend initially running WBs with each antibody separately so you can get a feel for the specific and non-specific binding characteristics. An alternative approach would be to cut your membranes so that each cut membrane only contains the molecular weight protein of interest and then do single Ab WBs on each. Another alternate approach would be to run one WB with one Ab, then strip the membrane and re-probe with your next Ab. Then repeat for the third. I've successfully used this approach many times with good results (although stripping the membrane will reduce some of your protein content and could affect the subsequent Ab binding; so you need to consider that). As I suggested above, you should run individual WBs for each Ab initially until you fully understand the properties of the Ab.
I hope that helps.
Up to three protein within one staining.
First recommendation for direct simultaneous stain on one membrane:
select antibodies with different enzyme labels: one with HRP, one with ß-Galactosidase, one with AP. For all three enzymes are differently colored substrates available.
The simple method: Run one gel with one small slot (for the MW standards) and one large slot for your protein mixture. After transfer you can make a stain with Ponceau S (which is removable in a simple water bath). You can make a scan or a copy of this protein stain. Knowing the the protein bands are you can cut your membrane into small stripes and now you can stain them differently, one for e.i. high sensitive protein stain (Aurogold, ...), the next with antibody one, two ...
After all this, put the stripe puzzle together and you can have an overview which proteins are present.
Some antibodies are quite dirty and light up bands in addition to their antigen. You need to check your antibody quality first to make sure that non-specific bands don't overlap.
Once you check this, you are free to combine as many antibodies as you want, though I personally have never tried more than two simultaneously.
LiCOR makes a system where secondary antibodies are labeled with red or green fluorophores and then you scan the blot to visualize your bands. This is useful because if your two primary antibodies are from different species (i.e. rabbit and mouse) you can use red-anti-mouse and green-anti-rabbit to see both simultaneously and you'll know for sure which signal came from which antibody.
And I believe quantitative PCR *IS* real time PCR. RT can mean reverse transcriptase (looking at mRNA) or real time. I know you can probe at least two PCR products in multiplex with qPCR using Taqman probes with different fluorophores - I've not tried more, but I imagine it's possible. You just need to make sure that 1) your fluorophores don't overlap, 2) your primers and probes are VERY specific and 3) your PCR reactions occur efficiently in multiplex. These are all things you just have to test for and find the right conditions, primer/probe ratios, etc- there's no good way to predict.
Feel free to contact me if you have questions about what I've written. Good luck!
Antibody specificity is really important for this type of blot! You would need to run all the controls (each primary Ab separately, in various combinations, secondary-only controls, etc) to be certain there is no crossreactivity. Becton Dickinson used to offer a service called "BD PowerBlot" that did this type of detection. They used infrared fluorescence to increase the amount of information from each blot. This publication used the PowerBlot approach:
http://cancerres.aacrjournals.org/content/66/23/11194.long
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