5 replies to this topic

[thedaywemet]

Hello,

I cloned my cDNA of interest into CMV-14 vector (3x flag, C-term tagging, Sigma)
I am trying to confirm the proper expression of my cloned cDNA in two ways. 1) immuno cytochemistry 2) western
My protein normally locates in the mitochondria, and I could detect it in mitochondria by immunocytochemistry.
But, in western blot, I detected 4 bands...I could not see these 4 bands in mock transfected or nothing transfected cells.
I sequenced the cDNA, and no error in PCR was made during the cloning step...
I wonder why 4 bands appeared in transfected lanes, and no non-specific bands in control lanes..
I heard sometimes the sigma flag antibody shows non-specific bands,
but in my case, those 4 bands were not detected in control lanes, which means it's not non-specific
Can someone please.. please.. help me??

The MTS signal locates at the very first region of the gene, that's right after the first methionine.
There are 4 more methionines after the first one, but.. I inserted Kozac (ACCATG) to ensure it starts from the first ATG.
Furthermore, I could detect the protein by cytochemistry, and it was in mitochondria, which means the translation really started from the beginning (including MTS which is just after the first ATG)

I will be so much grateful if you could help me!

[epibio]

What type of antibody are you using for the Western? You could be picking up degradation products of your expressed protein.

[Vini]

epibio, on Apr 20 2010, 12:26 AM, said:

What type of antibody are you using for the Western? You could be picking up degradation products of your expressed protein.

yeah, could it be possible that ur protein is getting chopped inside mitochondria (the MTS will be removed once inside, right?   ), or maybe during lysate preparation ur protein is getting degraded............also, what are the band sizes???

is the antibody against the flag???

Edited by DRN, 20 April 2010 - 06:20 AM.

[thedaywemet]

DRN, on Apr 20 2010, 11:19 PM, said:

epibio, on Apr 20 2010, 12:26 AM, said:

What type of antibody are you using for the Western? You could be picking up degradation products of your expressed protein.

yeah, could it be possible that ur protein is getting chopped inside mitochondria (the MTS will be removed once inside, right?   ), or maybe during lysate preparation ur protein is getting degraded............also, what are the band sizes???

is the antibody against the flag???

yes, the ab is aginast flag (sigma)
the endogenous protein (without flag tagging) is about 20kDa
2 bands are between 26 and 18, and other two badns are between 26 and 37.8kDa
1 band can be degraded one, but at least one band become bigger...how much weight does it increase if it's phosphorylated, btw??

[Vini]

thedaywemet, on Apr 21 2010, 02:42 PM, said:

DRN, on Apr 20 2010, 11:19 PM, said:

epibio, on Apr 20 2010, 12:26 AM, said:

What type of antibody are you using for the Western? You could be picking up degradation products of your expressed protein.

yeah, could it be possible that ur protein is getting chopped inside mitochondria (the MTS will be removed once inside, right?   ), or maybe during lysate preparation ur protein is getting degraded............also, what are the band sizes???

is the antibody against the flag???

yes, the ab is aginast flag (sigma)
the endogenous protein (without flag tagging) is about 20kDa
2 bands are between 26 and 18, and other two badns are between 26 and 37.8kDa
1 band can be degraded one, but at least one band become bigger...how much weight does it increase if it's phosphorylated, btw??

one unit of phosphorylation will lead to an increase by 80 Da, but there could be more than one phosphorylations...........also it could be some other PTM, like glycosylation.  However, the mobility of the protein may be shifted due to these PTMs and its not necessary that mobility strictly follows the additional mass........

also, i dunno abt the size of the tag that you have used, but could it be that the lower band corresponds to just the tag???

[thedaywemet]

I was thinking about the possibility of PTM, too..
Now I transfected the plasmid into other type of cell line and trying to sea the band pattern if it shows the same thing.
Thank you for your help!