Frequently Asked Questions
► What is the difference between Proteofectene® and Proteofectene®AB?
► What kind of quality must have used protein or antibody for a Proteofection?
Any impurities, contaminants or additives present with your protein or antibody of interest may affect delivery efficiency PROTEOfectene® oder PROTEOfectene® AB. Consequently, use a protein/antibody as pure as possible.
Stabilizers such as detergents can inhibit the delivery if present excessively in comparison to the protein of interest. Stabilizers such as glycerol or other similar additives do not interfere with the protein delivery.
► Does BSA interfere with the Proteofection with Proteofectene AB?
BSA in the antibody solution inhibits antibody delivery! 0.1% to 2% BSA are common concentrations in antibody solutions – this means 1 mg/ml to 20 mg/ml. The proportion of BSA greatly exceeds the antibody in these solutions. BSA competes with the antibody during the formation of the antibody–PROTEOfectene® AB–complex and therefore inhibits antibody delivery.
► Do preservatives interfere with the Proteofection?
Preservatives such as sodium azide could hypothetically lead to cytotoxicity if present in high concentrations. It can be removed by dialysis if necessary.
► What is the Proteofectene® Reagent and how does it work?
PROTEOfectene® is a lipid-based formulation which forms non-covalent complexes with proteins. These complexes are internalized by cells and the proteins are released into the cytoplasm.
Just incubate your protein of interest with PROTEOfectene® in 1xPBS for 10-15 min, dilute with serum-free medium then apply the mixture to your cells and 3-48 h later, you are ready to assay for protein activity.
► The proteofection of my cells with Proteofectene® and the positive control R–Phycoerythrin is successful, but the proteofection with my protein doesn't work!
Due to their specific properties some proteins might not be efficiently delivered with PROTEOfectene®. So, basic proteins, which are positively charged under physiological conditions are very difficult to introduce into cells. Another reason could be impurities of the preotein (e.g. detergents, preservatives, stabilizers).
► Why proteofect purified proteins directly?
Protein transfection (Proteofection) is extremely rapid compared to gene expression studies using transfected DNA, because it bypasses cellular processes such as transcription and translation. In addition potentially harmful random DNA integration into the genome of the target cells is avoided. PROTEOfectene® makes it possible to deliver active proteins directly into cells for studies involving all kinds of questions regarding cell cycle, apoptosis and many other factors.
► Why is R–Phycoerythrin included in the Proteofectene® kit?
R–Phycoerythrin (100 µg/ml) is provided with PROTEOfectene® as a positive control. Use it with a protein:lipid ratio of 1:2 (for 1 μg of protein 2 μl of PROTEOfectene® are needed).
R Phycoerythrin has been delivered into many different cells, the probability of a successful transport is therefore high. This control protein is provided to help you set up your experiment and should be used for each new cell line with which you experiment. If cytotoxicity is a problem, the positive control enables the user to determine whether the delivered protein is influencing cell viability.
► Why is FITC–IgG included in the Proteofectene®AB kit?
FITC–labeled IgG (positive control, 100 μg/ml) is a fluorescence–labeled antibody (immunoglobulin) as a positive control. Use it with a antibody:reagent ratio of 1:1 – 1:2 (for 1 μg of antibody 1-2 μl of PROTEOfectene® AB are needed). This control antibody is provided to help you set up your experiment and should be used for each new cell line with which you experiment. FITC–IgG has been delivered into many different cells; the probability of a successful transport is therefore high.