hi i am really confused here. so let me describe what my experiments is all about. I want to look at whether certain signal pathways are activated upon mechanical stress onto cells, so i exposed my cells to mechanical stress at duration 0 min, 10 min, 20 min etc. from the protein lysate, i ran it on gel and transfer onto a membrane. Then i probe using antibody to detect phosphorylated form of signal protein. I then strip antibody, and detect using antibody against phosphorylated and unphosphorylated form (total signal protein) of protein.
so this is considered as 1 replicate. i repeated and obtained 2 more biological replicated. for each one of them, i calculated the ratio of phospho to total protein. i then normalize the ratio within each blot to their respective controls (0 minute mechanical stress).
i made a silly mistake of not sticking to a particular exposure timing when i expose my blots. i was wondering if i construct the graph using the normalized values, will the values 'add up'? that is, is it comparable? or is it not comparable simply because i didnt stick to particular exposure timing.
Often, there are too many replicates to fit on a single gel. Moreover transfer is rarely 100% and is an uncontrollable variable in every blot, even if loading carefully, treating with substrate, and exposing is held "constant". I like to normalize each phospho- and total- band signal for loading/transfer variablilty by dividing by each lanes total protein signal (all bands stained by a generic protein stain such as Ponceau or Coomassie). Then for each blot, I normalize all lanes of a given band by divding each by the total signal from them all. This brings each lane to a < 1.0 value, which can then be compared with those from other blots without concern for variation in loading, transfer, substrate treatment, exposure, etcetera. In essence, total and phospho signals should be normalized within the blot before looking at their ratio.
- Badges
- Science method