Dear Heba 

The expression level of caspase 8 dependes on the cells' condition, because as you know it increases markedly during apoptosis. Cyclin D is related with mitosis, and particularly with one phase of it. So that precisely the name cyclin comes from the fact that its level is "cyclically" modified. P 53 increases under stress conditions.

Therefore it is not possible to establish a constant level for these 3 proteins because they change along the cell's life cycle.

Best regards

Tomas 

Tomas has given you a good answer, and the answer is how long is a piece of string!  I found when working with new proteins find a good positive and a good negative control, which you can find in the literature, and I am sure that your cells will be somewhere in the middle.  With p53 you need to ascertain whether it has wild type p53, and a good way is to irradiate them in the presence and absence of nocodozol and analyse by FACS, again this is quite commonplace and there are methods in the literature.  Cyclin D ( you did not say which one you are looking at, and they are quite different) generally is induced by mitogens, so serum deprive you cells and then re-stimulate with 20% serum and do a time course of expression.

Good luck!

Hi Heba,

As it was already said it is not possible to establish an expression level for these 3 proteins because it depends on the cell model you are analyzing and other conditions. However, you don´t need to know these information to proceed with the western, if your cells are adherent, you can seed then in a regular dish and allow then to grow moreorless for 3 days until they reach around 70% of confluence. Generally, for a 50 cm2 dish around 50ml of lysis buffer is used. Normally, 30-60 microgramas of the cell lysates per well in the gel is sufficient to see the expression of most of the proteins if they are expressed.

Good luck

Hi Heba,

if you are working with cancer cells, it is perfectly possibile that either p53 or caspase-8 are not expressed. TP53 is one of the most frequently mutated genes in cancer, with point mutatons, frameshift insertions or deletions which can result in the absence of the protein or the expression of a truncated form. Caspase-8 could be lost in some tumors, for example in certain neuroblastoma subgroups. So, I suggest to carefully read the literature or public genomic data bases before starting with your experiments: in the case of cell lines, you should be able to easily retrieve information about p53 expression levels and mutations, and get prepared in case the MW of the protein is not 53 KDa or your WB is blank. It is also important to check which p53 epitope your antibody recognizes. Anyway, always use positive reference controls in your WBs!

For cyclin D (D1, I suppose?), the situation is very different: it is expressed during G1 phase and persists during the cell cycle up to the end of mitosis. If your cell culture is exponentially growing, it should be easily detectable by WB like a housekeeping protein. Moreover, in cancer cells cyclin D1 is frequently overexpressed due to multiple mechanisms. So, in the case your WB signal is too low, I suggest to play with technical variables to increase sensitivity (type of primary Ab, primary and secondary Ab concentrations, detection method -ECL is more sensitive than fluorescence- and so on). And remember to add positive controls! :-)

I hope this helps, 

Fabio