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SAINT-PhD efficiently delivers proteins into mammalian cells  

The improved SAINT-PhD protein delivery reagent is ideally suited for the non-cytotoxic
delivery of biologically active cargo molecules, such as proteins, antibodies or small
peptides, directly into living cells.

This unique reagent is an effective tool for pathway research and analysis, target validation
and protein or peptide library screening. The SAINT molecules have been put into a new
formulation optimized for a fast and easy method to study protein function on a cellular level
directly. This approach avoids the problems of DNA transfections such as promoter interference
and unpredictable levels of transfection in particular cells.

How does it work?
The freshly prepared complex of SAINT-PhD and the attached cargo protein will translocate itself
into cultured cells when added directly to the culture medium.

Preparing the complex of SAINT-PhD with cargo
molecules is a simple process diluting
appropriate amounts of your protein with the
supplied hepes buffered saline (HBS)
and adding the necessary volume of SAINT-PhD.

 

The complex will be formed immediately and can be added directly to the cultured
cells. If necessary using your specific and personalized protocol the complex can also be
diluted with fresh culture medium.

This improved reagent presents significant advantages which include the delivery of antibodies
into cells in less than 4 hours, its relative ease of use and its serum compatibility during
the protein delivery.

A range of different cell types have been tested with SAINT-PhD. Protein delivery efficiencies
of up to 100% of the cells have been reached. The cells that successfully have been tested
with SAINT-PhD are listed below:

Adherent cell lines:

- B16F10
- COS-7
- HEK-293A
- SKOV-3
- U373MG
- 3T3
- CHO-K1

Adherent primary cells:

- HUVEC
- Human Dermal fibroblasts

Suspension cell lines:

- Jurkat
- HeLaS3

 

In all of these cell lines, SAINT-PhD is a very effective intracellular delivery vehicle
for proteins. Some of our observations:

Very high uptake percentage.
Up to 100% of all cells exposed to the protein-SAINT complex show internalization of
the protein (see also Fig 1).

 


Figure 1: RPE-labeled mouse IgG was mixed with SAINT-PhD (20L) and incubated for 4 hours
with SKOV-3 cells in a 24-well plate. Untreated cells (only SAINT-PhD added) are shown in A.
The RPE-labeled antibody was delivered efficiently as 0.1g is already delivered in 35 percent
of the cells (B). Adding 0.25g (C), 0.5g (D), or 1 g (E) did increase the efficiency up
to 100% respectively, while 2g (F) delivery showed an increased mean fluorescent intensity (data not shown).

 

Linearity.
The uptake of protein is linear with the amount of protein added to the cell culture (Fig. 2 and 3).

 


Figure 2: Linear correlation of delivered vs. complexed IgG: FITC-labeled IgG ( 10 ng 300 ng ) was
complexed with 5 L of SAINT-PhD. Fluorescence intensity of the gated cells correlated with amount
of protein to be delivered. Controls without SAINT-PhD showed no fluorescence.

 


Figure 3: Uptake of -galactosidase by B16F10 Cells. -Galactosidase (1ng to 1000ng) was complexed
with 5L of SAINT-PhD, and added toB16F10 cells in a 96-well plate. After 4 hours cells were stained with
X-gal. Color intensity was directly proportional to amount of -galactosidase added.

 

Functionality preserved.
After delivery, the protein has not lost its activity. For example, -galactosidase retains it enzymatic
activity after uptake (Fig. 4).

 


Figure 4: Uptake of -galactosidase by CHO-K1 Cells. 1g was complexed with 20L of SAINT-PhD. The
complex was delivered in a 24-well plate, incubated for 4 hours and stained with X-gal.

 

No effect by serum.
The presence of serum in the cell culture medium does not affect the rate or level of uptake of protein by SAINT.

 

Complete cell viability.
In contrast to most other lipid formulations, cell viability is excellent. We observe often >99% viability,
even after 24 to 48 hours of exposure without removing the complex.

 

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Copyright 2002 GENTAUR Molecular Products
Last modified: 05/29/09