I would like to measure the expression of several proteins or mRNA simultaneously. I mean, can I measure by one nitrocellulose membrane (as all of my target proteins or mRNA will be present there) in the case of WB or RT-PCR? Or I have to prepare separate WB/ RT-PCR for each of the proteins or mRNA? I would like to use several primary antibody to detect my target proteins from one nitrocellulose membrane. I think, the commercial suppliers use same Fc portion for primary antibody so that they can be detected by same secondary antibody. If so, can I use several primary antibody and then the secondary antibody to detect and measure all my target proteins together? It will be easier to compare all the expressions by a single WB/ RT-PCR. Can I?
You can use several primary antibodies at once in a Western Blot but you must be sure that they are really specific and don´t cross react. If not you won´t be able to analyse the blot due to several wrong signals. An alternative would be to use bigger slots in your gel and cut the blot afterwards in strips which are incubated with the single antibodies.
Parallel quantification of mRNA can be done by specific TaqMan probes labelled with different markers (FAM, HEX, TET, ROX...) for each target but not with SYBR green.
You can use several primary antibodies at once in a Western Blot but you must be sure that they are really specific and don´t cross react. If not you won´t be able to analyse the blot due to several wrong signals. An alternative would be to use bigger slots in your gel and cut the blot afterwards in strips which are incubated with the single antibodies.
Parallel quantification of mRNA can be done by specific TaqMan probes labelled with different markers (FAM, HEX, TET, ROX...) for each target but not with SYBR green.
You can detect different proteins on a single blot using multiple primary antibodies, but you cannot compare their levels unless you have also run standard curves with known amounts of each target protein. The reason is that the different antibodies will have different affinities for their targets.
Thank you very much. That means, its better to detect by several WB? What about RT-PCR? Is it possible to use several primers together to multiply several mRNAs?
You can use multiplex fluorescence to detect more than one protein per blot.
In general, it's better if you can detect and compare the proteins (relative quantification) from the same blot. To compare multiple blots, you will need accurate standard curves. The gel loading and transfer process introduces a LOT of variability, making blot-to-blot comparisons trickier and less accurate.
In addition to everyone's great suggestions, you may want to consider commercially available protein array. You can probably get a custom array if you have sufficient number of proteins you want to look at. If it is just a handful of proteins then all the suggestions above would suffice.
For RNA, multiplex assays (some already mentioned) would be the way to go. One multiplex assay you can consider is digital molecular barcoding technology, available from Nanostring. We had some samples submitted and looked at 12 transcripts, but you can look at hundreds at one time if that is what you are after. Alternatively, you can consider RNase Protection Assay (RPA). I don't know if there is still a commercial source for RPA reagents, but many labs around the world have the DNA constructs you would need, perhaps consider requesting from them. It is a laborious assay, but relatively inexpensive if you already have the constructs from which you generate your radiolabeled probes.
Links below for articles describing the RPA assay (they are in mouse, but human probes exist as well).
http://www.ncbi.nlm.nih.gov/pubmed/16000858
http://www.ncbi.nlm.nih.gov/pubmed/9328562
- Badges
- Science topic