ProteoCarry (Protein Transfection Reagent)

Product#: FDV-0015
$400.00
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ProteoCarry (Protein Transfection Reagent) 

Cat.No. 
FDV-0015
Size  1 Set
Store at -20°C


Description

ProteoCarry is a new type peptide base transfection reagent.
When using a conventional protein transfection reagent, transfected proteins are accumulated in endosome or lysosome, and most of such proteins are dissociated in lysosome proteolysis system. Contrary, ProteoCarry can deliver transfected proteins to cytoplasm effectively. Moreover, pre-incubation with proteins are NOT needed. Therefore effect to the protein functions can be minimized.


Product Background

ProteoCarry is a novel peptide-based transfection reagent for exogenous proteins such as functional proteins and antibodies with highly efficiency of cytosolic delivery. Protein transfection, intracellular delivery of proteins, is a powerful method to analyze cellular response to the protein of interest. While plasmid-based gene expression generally needs 12-24 hours to express proteins well, protein transfection reagents can immediately import functional proteins into living cells within several hours.

Although many protein transfection reagents including peptide-, cationic lipid- and polymer-based compounds have been developed to date, these reagents struggle to deliver proteins into “cytosol”. Commonly these reagents interact with
protein to form complexes and subsequently protein-reagent complexes are entered into cells via endocytosis pathway. But protein-reagent complexes are able to escape endosomes with little efficiency and consequently transported to lysosomes to be degraded. Highly effective endosomal escape of proteins is the most important subject of protein transfection reagents.

ProteoCarry can overcome this problem based on a novel and potent pHdependent endosomal membrane-lytic activity. Proteins and ProteoCarry can be delivered into endosomes by endocytosis pathway and also escape to cytosol by its highly membrane-lytic activity with low cytotoxicity.

Features

  • Highly soluble in water
  • High efficiency of delivering proteins to cytoplasm
  • Fast - 1 hour incubation
  • No pre-incubation with proteins needed
  • Effect to the function of proteins cab be minimized
  • Low cytotoxicity
  • Polysaccharide can be also transfected.

Kit components

  • ProteoCarry, 4mg in vial (vial A)
  • FITC-dextran as positive control, 2 mg in vial (vial B)

Storage

  • Shipping : Room temperature
  • Store : -20℃
Validated cell types
  • HeLa, SW280, COS7, NIH3T3, HUVEC<>li/
Procedure overview
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About protein transfection reagent and issue of conventional one
Protein transfection reagent has been widely used as it can deliver proteins faster than inducing proteins by gene transfer.
Many reagents have been developed so far, such as peptide-base, cationic lipid-base, and polymer base. Proteins transfected by such reagents generally form complex with proteins and incorporated to cells by endocytosis. After that, the complex breaks endosome membrane and transported to cytoplasm. 
 
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Figure 1. The principle of ProteoCarry
Left: Problems of conventional competitors    Right : Principle of ProteoCarry

However, current these reagents have weak activity for disrupting membranes.
Therefore, such arrested proteins cannot escape from endosome. Arrested proteins are considered to move to lysosome proteolysis system.
Therefore, transfection reagents should have strong activity for disrupting membranes so that transfected proteins could be functioned and transported in cytoplasm effectively.

ProteoCarry, a new peptide-base transfection reagent has a strong and selective membrane-disrupting activity. ProteoCarry does not require a forming of protein-reagent complex. Therefore, not only proteins, but also biopolymers can also be transfected to cells.

Preparation of 50x ProteoCarry stock solution
  1. Add 581 µl of sterile ultrapure water to vial A and mix gently well to be 50x concentrated solution.
  2. Stock solution should be stored at -20oC for up to 6 months in small aliquots to avoid repeated freeze and thaw cycles.
Preparation of positive control
  1. Add 1 mL of sterile PBS to vial B and mix gently well to prepare 10x stock solution (2 mg/ml).
  2. Stock solution should be stored at -20oC.
Preparation of protein solution
  1. Prepare 10x concentrated protein solution with sterile PBS. 10x solution should be prepared at time of use.
  2. If it is challenging to prepare 10x concentrated solution, please optimize Table 2 below

 

Final conc.

10x conc.

Antibody

50-250 µg/ml

500-2500 µg/ml

Proteins

 

 

  • Saponin

1-10 µg/ml

10-100 µg/ml

  • Cre

10-100 µg/ml

100-1000 µg/ml

Positive control

 

 

  • FITC- dextran

200 µg/ml

2 µg/ml

Table 1 Suggested concentration of proteins
Memo
To observe cytosolic disperse of fluorescent signal from fluorophore-labelled proteins such as FITCIgG, high concentration of proteins may require. Under the low concentration of fluorescent proteins,
fluorescent signal from endosome may be higher than the signal from cytosol.

Transfection protocol
CAUTION
Following protocol is an example for the positive control experiment, FITC-dextran.
Transfection efficiency is highly affected by protein’s profile such as molecular weight and net charge, celltype, and confluency of cell. To obtain best results, please optimize condition for your experiments.
Cell preparation
Cells are seeded and allowed to reach up to 80% confluency in 24 hours.
Memo
Confluency of cultured cells may be influence on transfection efficiency. Please optimize cell
number for each experiment.
Transfection
1- Mix reagents according to the following table (= Transfection mixture).
 

Scale

50x ProteoCarry(µL)

10x Protein soln (µL)

Base medium volume (µL)

Total volume (µL)

96 well

2

10

88

100

24 well

10

50

440

500

12 well

20

100

880

1000

6 well

40

200

1760

2000

Table 2 Suggested amount of transfection mixture
 
Memo
You can choice serum-free, serum-supplemented media or PBS as the basal medium of protein
transfection. Delivery efficiency through ProteoCarry is not affected by serum (<10% FBS). In some
cases, PBS give the best results. Please select a basal medium according to your cells of tested. 
2- Remove culture medium, wash cells with PBS twice and add the transfection mixture prepared above.
3- Culture cells in the transfection mixture at 37oC for at least 1 hour. The incubation time for the best delivery efficiency is depends on proteins. Please optimize incubation time.
4- Remove culture medium, wash cells with PBS twice and add fresh medium containing serum and culture cells for appropriate time for your experiments. Cells can be used for following applications

Memo
Mechanism of intracellular delivery of proteins by ProteoCarry is based on endocytosis and
macropinocytosis pathways. Please avoid to use inhibitors for endocytosis and/or macropinocytosis
pathway during transfection.

Application data

1. FITC-dextran (positive control)

Cell: HeLa (80% confluency)

Cargo : FITC-dextran (200  mg/ml)

ProteoCarry : 1x concentration

Transfection time : 1 hour

2. Fluorofore-labeled IgG

Cell: HeLa (80% confluency)

Cargo : Fluorofore-labeled IgG (250 ?g/ml)

ProteoCarry : 1x concentration

Transfection time : 1 hour

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