I need to analyze a specific intracellular protein level in control and infected human monocyte-derived dendritic cells
I think the perfect way at the first step is purify your pro then use methods like HPLC or etc.
Hi Amanda, I think doing HPLC/FPLC just to check protein expression levels is a bit unnecessarily complicated. You can try SDS-PAGE to start, just make sure you load equal amounts of total protein in the gel for each sample. For Coomassie blue detection (which is done before WB), 50-100ng total protein should be enough. You can then assess by eye if your expected protein is more/less abundant versus the control.
After SDS-PAGE you can go to either WB or ELISA if you want to be very specific.
Hope this helps,
Andrew
I agree with Andrew Michael Hogan . It also depends on how much of the protein you have. If you expect to have a lot, you could start with SDS-PAGE and western blot. If not, try ELISA or flow cytometry. ELISA would let you do ng/mL while with Flow, you can only see if more of your cells are expressing a particular protein. You could use MFI to see if there is a difference in the expression level but ELISA is better when looking at quantities.
Western blots are at best a semi-quantitative technique. ELISA is simpler and more reliable, but in my experience dot blots are even better. You simply filter your protein solution over a PVDF membrane in a 96-well vacuum manifold. Then develop like a western. The PVDF membrane will bind more than 95% of your protein, while binding to a plate takes longer and has an efficiency of only a few percent. If you use electronic chemiluminescence detection, dot blots are linear over at least 5 orders of magnitude (ELISA only one order).
Engelbert Buxbaum Thanks for share this matter.
Andrew Michael Hogan
I think that the SDS-PAGE to start is right.
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