Dear All,
I have question on choosing WB bands image for publication. My target protein has two forms: 1.full-length form (very strong signal) 2. cleaved form (faint/low signal) in the same blot. In case to present both band in the same image, the cleaved band has to be exposured for longer time to have clear/sharp band, however, it will make full-length band oversaturated. If I have shorter time exposure (same blot), the full-length band will not saturated, but the cleaved band will be very faint (hard to see).
In this case, Should I seperated these two bands from the same blot into each individual image to have clear/sharp and not saturated for each band (shorter-exposure image for full-length, and longer-exposure image for cleaved form, but both are the same blot)?
*In density quantification, I choose unsaturated band from different exposure time images to calculate.
Thank you
Cheerio,
Hi Asa,
You certainly have to present a full blot (and unsaturated) showing the full length protein and the cleavage product as it is what you are using to make your quantification. And even though the cleavage product is weak, it simply represent the reality (i.e. weak cleavage).
I meant you should have on the same blot the full length AND the cleavage product - it is important to show how much cleavage product you get compared to the total amount of full-length protein, especially since you are quantifying the cleavage.
Make sure you are focusing on the right problem as what you really want to show in the end is that putative cleavage is specific. Can you find experimental conditions to prevent this cleavage? Or in contrast increase it? By doing so you will solve your initial problem and also strengthen your observation.
- Badges
- Science method