[Cytometry] Quantitation of Total Protein Content by Flow

Howard Shapiro hms at shapirolab.com
Sun Feb 1 12:14:10 EST 2009


Jodi Moore wrote-
> I was wondering if anyone could suggest a dye or dyes that I could use 
> to quantitate total cellular protein content in unfixed cells. The 
> lasers I have at my disposal are a 405nm, 488nm, 594nm, and 633nm.  
> Ideally, we would want to use this dye in combination with a viable 
> DNA dye (i.e. One of the Vybrant Dye Cycle reagents) on cells that 
> would be sorted and then put back in culture.
> We are also looking to use SR101 in combination with DAPI, AF488 and 
> AF594 on fixed cells. Any recommendations/protocols/tips would be 
> deeply appreciated.
>   
The only dye that I am aware has been used for cytometry of total 
protein in unfixed cells is rhodamine 640, which is the same dye as 
rhodamine 101. It will excite very well at 594 nm and acceptably at 488 
nm. The references, both available free online from the journal web 
sites, are:

Crissman HA, Steinkamp JA. Rapid, one step staining procedures for 
analysis of cellular DNA and protein by
single and dual laser flow cytometry. Cytometry. 1982; 3:84-90.

and

Steinkamp JA, Orlicky DA, Crissman HA. Dual-laser flow cytometry of 
single mammalian cells. J Histochem Cytochem. 1979; 27:273-6.

SR101 has the same spectrum as rhodamine 101/640, which means that it 
can't be used with AF594-labeled antibodies or other ligands, because 
AF594 has a very similar spectrum. It should be compatible with 
phycoerythrin or with a red-excited label. It also works fine with 
pyronin Y (measured in the PE channel), allowing DNA/RNA/total protein 
measurement to be done in unfixed cells in a 2-laser instrument.

In some situations, notably bacteria and peripheral blood leukocytes, 
good correlation has been observed between side scatter and total 
protein measurements; your mileage may vary.

-Howard


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