Re: Protein Content

Andrew Beernink wrote:

>
> Thanks  [to Ultan Cronin] for your detailed response.  I failed to  
> clarify that I was looking to quantify protein content "within" the  
> cell cycle, i.e. G0/G1, S, G2/M.  I'm aware of the uses of CFSE for  
> tracking cell divisions; unfortunately, I'm unaware of how to  
> properly phrase a question to the Purdue list!
>
> I know that Pyronin Y is used for this application.

Not for protein content; pyronin Y, when applied in conjunction with  
DNA dyes such as Hoechst 33342, stains double-stranded RNA. The  
Hoechst/pyronin staining pattern will discriminate cells in G0 from  
cells in G1 and also identify both proliferating and quiescent cells  
in S, G2, and M (quiescence in these cell cycle phases can occur,  
e.g., in tumors).

> I'm simply wondering, based on the huge availability of fluorescent  
> dyes as succinimidyl esters, whether anybody had tried to duplicate  
> early efforts to quantify protein using isothiocyanates like FITC  
> (as per the paper I noted).
>

CFSE will work provided you don't leave stained cells around long  
enough for them to divide.

Most acid dyes, including fluorescein, sulforhodamine, etc., will  
stain total protein in cells; Zbigniew Darzynkiewicz, Harry Crissman,  
John Steinkamp et al combined FITC with Hoechst/pyronin decades ago  
to follow DNA, RNA, and protein through the cell cycle. FITC was used  
because the covalent staining eliminates the high background  
fluorescence typically encountered when using acid dyes as protein  
stains.

It was also noted that rhodamine 640 could be used in combination  
with Hoechst/pyronin for vital staining of protein.

There is some evidence that the side scatter signal provides much the  
same information as a total protein stain; this is definitely the  
case with bacteria and usually so for peripheral blood leukocytes.

All that said, you could pretty much use any protein-reactive dye as  
a "total protein" stain, giving you considerable flexibility in  
choice of spectral characteristics. The "early efforts" haven't been  
duplicated because most people are not terribly interested in  
staining total protein, not because there are significant problems  
with old methods.

-Howard



-- End --