According to my experience,  when you have to quantify the cleavage of a protein is always better to detect the disappearing of the full length protein rather than the appearing of cleaved bands. The cleaved bands can be further degraded by non-specific proteases making the quantification difficult. Check for example my paper on Scythe cleavage by caspase 3 (PMID:22285488)

Cheers,

Giulio

If the two bands are detected with the same sensitivity, then you could add up the intensities of the bands to get the total amount of protein.

As Guilio already recommended it is advisable to determine the differences in the amount of the total protein as antibodies may less efficiently if at all recognize the protein fragments. However, to determine differences reliable application of equal sample amounts is necessary. Therefore, a concomitant stain of a "house-keeping" protein which is constantly expressed in the tissue/cells is useful. So you can normalize your signals on those of the "house-keeper".

Best wishes, Peter

Are you working with the cell-free extract or a purified protein, Mazhar?!

Thank you Giulio for your paper. I had downloaded few days before and checked it. It really helped me in determining what I need to do. But, I am still in trans because, in your case, your cleaved bad was indicator of the Caspase-3 activity. But in my case, I am concerned with the overall expression of the protein. Although, I am still confused, but caspase is really relevant to me. It will help me in future.

Thank you Adam for your  response. Your answer also seems logical because if a protein is stable in two states, then it might be a good idea to quantify both of them. But, I am still looking for valid scientific grounds to put my quantifications on them.

Thank you Peter, for acting as a tie-breaker between Giulio and Adam. and yes I am using actin as a control and I am comparing my lanes with their own respective internal controls. But, I struck at the point when I observed this double band. 

Natalia, thank you for your comment. I am working on MDCK cell lysates. I extract protein from the cells and check the expression of the protein. Any suggestions from you are also welcomed. let me know, if any information necessary about my experimental design is required.

Regards

It is not clear from the description what is the goal of your experiment. Do you study the expression of specific cell protein upon viral infection or you suspect that occasional  viral infection of culture occurred during protein induction so the protein of interest is cleaved? I would check the specificity of your primary antibodies with serially diluted purified  target protein (of course if it available). In my experience  an observation of  two bands on WB in control untreated cell lysate may indicate on non-specific protein recognition by used antibodies. If used antibodies are specific to target protein and the second band belongs to cleaved target protein you should add protease inhibitors to the extraction buffer. There are commercial Protease Inhibitors cocktails from Clontech, Sigma Aldrich etc which work pretty well suppression of different classes of cell proteases...

If, after you have established that the proteolytic cleavage did not occur during sample preparation, then as suggested above, you will want to get some measure of how your antibodies bind to the cleaved bands compared to their binding of the intact protein. May I suggest loading increasing or decreasing amounts of your sample (about 5 or 6 different amounts where the western blot intensities would be linearly related to the loading amounts. It does not have to be purified protein. Just make the required dilutions from the same exact sample. Then after running the western blot, plot band intensities vs amount loaded for each size. If you get the same slope for the three plots, then the cleaved bands are more or less binding the antibodies in the same way. If only one of the cleaved bands gives the same slope as the intact protein band, then you would use that data plus the data on the intact protein for your experiment. 

Hi everyone, following is my blot,

from left to right

is Ladder, control A, Control B, Infection MOI1, Infection MOI3, Infection MOI5 in Virus Challenge.tif

and

from left to right 

is Ladder, 4ug, 8ug , 15ug ,23ug of cell total protein (Antibody Titration) and I am using 20ug protein

So, let me address all queries one by one:

1) the antibody is very specific, I have done titration of the antibody previously. What happens that on the infection, I observe a clear shift in the band to lower molecular weight and sometime a very faint band remain at the proper position (compared to control A, control B). 

2) What I am trying to determine? I am trying to determine the change in the protein expression when cell is challenged with the virus. 

3) Regarding protease inhibitor? I am already using protease inhibitor cocktail while lysing my cells and extraction of protein. Everyone in the lab is using same protocol and we have very fine results. And our western blots are also very good.

4) Regarding Antibody titrations: yes I have done antibody titration and I am already using optimized concentration of protein and antibody dilution.

Please let me know, if any further information is required

Regards

Mazhar, Are you referring to the band that is just below the mol wt marker seen in lane 1? As I understand it, you want to compare the amount of polypeptide made depending on viral challenge. I would suggest that you lower the amount of protein that you load into the first gel. This is because in the second gel, the one with different amounts of total cell protein, the intensities of the main band don't seem to change from the load of 15 to 23 ug. You want to be loading an amount where a plot of band intensity vs load is close to linear. So here is what I would suggest. (1) Plot band intensity from gel 2 of the main band vs load. What is the linear region? Determine the load to use for your first gel by choosing a load still within the linear region. My guess is that you will end up with a number that is closer to 15 ug than to 20 ug. With such a load, the band representing the cleaved fragment is of very low intensity. But if you are worried about it, then you could do something like this: (2) Repeat gel 2 but go on to higher loads. Your main band will be overloaded, but you will have better intensities for the cleaved fragment band. Plot band intensities of the cleavage fragment vs load. Again, check for the linear region and determine how much load to use to stay in the linear region but get visible bands. It would be best if this load is a simple multiple (for example, 2 or 3 times) of what you decided on in the previous step just to make it easier to conceptualize. (3) Now you can run the real experiment as in your first gel, but this time use the load determined in step (1). On the same gel, load your samples again but this time use the load you determined in step (2). Then you can compare how much main band was produced when challenged with different strains of virus. You can also compare how much of the cleaved fragment was produced when challenged with different strains of virus. I would keep the conclusions separate unless the antibody is commercial monoclonal antibody to just one epitope, then you can go further in your conclusion, but remember you used heavier loads when visualizing the lower mol wt band. Hope this helps!

Mazhar ,for quntification just i will recomend you ELISA techinique in your case because your western result show some degaradation of protein so total protein will be the sum of degraded and and intact protein.or you can try real time for quntification of expression of your geneof interest  and compare the expression profile from ELISA with real time PCR results it will give the clear picture of  your total expression of the specific gene.

Best Regard 

ISLAM KHAN

 M.SC (  BIOTECHNOLOGY )

M.PHIL (Molecularbiology)