I load 4ng and then 200ng protein in two adjacent wells. 4ng protein (conc. 0.8mg/ml) is derived from a two step serial dilution of 2mg/ml protein. Proteins are transferred to Fluorescent sensitive membrane. Overnight blocking is done at 4°C with Licor intercept blocking buffer and then incubated with primary ab (200ng/ml) and IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody (@1:10000 dilution) each for 1 hour with interim washes with PBST for 30 minutes. Image is captured at 800 channel., 169uM medium resolution.
Unfortunately, the signal from 4 ng band is inconsistent specially in Non-reduced blot. Whereas, theoretically the 4ng band should be 2% of 200ng band, practically I am getting signal
You could increase it simply by longer exposure time but because of a high background and more intense bands of the 200ng, this won't solve your problem. In fact, the main problem seems to be that you are expecting too much from the method.
The difference between the 200 ng and 4 ng is 50 fold. You need to consider the high background of this antibody which leads to a decreased dynamic range for your detection. Assuming a 16-bit image format of your scanner, you can only have ~65000 greyscale resolution. If you have already a high background (e.g. at 2% of the total maximum intensity) your real dynamic range will be less than ~50 (
LiCor imaging is super sensitive to background contamination in buffers, membranes, etc. Is your membrane validated for use on the Odyssey? Is your blocking buffer contaminated? (try BSA or fresh non-fat dry milk) Clean the glass panel on the scanner. Those look like streaky background blotches from insufficiently cleaned glass. Are you maintaining hydration on your membrane while imaging?
I am using Immobilon-Fl blotting paper and licor intercept blocking buffer (which is probably a fish serum buffer) which are recommended for fluorescent applications. Also, I clean the scanner with 70%EtOH and IPA and ensure a hydrated blot for imaging. Recently we could get rid of the streaky backgrounds after we started pressing the blot with roller before imaging. But that did not improve the signal though.
Miss, I totally agree with Huseyin. However, you can use PVDF membrane which has the higher protein binding capacity. For my fluorescent western blot, nitrocellulose membrane works fine. Alternatively, you could divide your gel into two by simply loading a marker in between and image them separately. I hope it helps!
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