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Lipodin‐Pro™ Experimental Results
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From Biology to Discovery™
Lipodin‐ProTM ‐ Protein Transfection Reagent
Cat. No 500100 Cat. No 500110
Description
The delivery of proteins inside living cells represents an alternative to nucleic acids transfection and a powerful strategy for functional studies or therapeutic approaches. Several technologies based on the use of peptide transduction domain (PTD) were developed successfully to transduce proteins across the plasma membrane. However, these PTD poorly interact with proteins, and covalent linkage between the protein and PTD is most often required. Lipodin‐Pro™ is a formulation of lipids able to capture proteins through electrostatic and hydrophobic interactions. There is no need for covalent linkage procedure, Lipodin‐Pro™ is directly mixed with the protein of interest for 10 minutes. The mixture is then added to the cells in culture, the lipid‐protein complexes are internalized by the cells and the proteins are released into the cytoplasm within few hours without any cytotoxicity. The optimized formulation of Lipodin‐Pro™ is fully biodegradable maintaining a high cell viability upon delivery. The proteins delivered inside the cells with Lipodin‐Pro™ retain both their structure and function whether peptides, proteins or antibodies are used.
1. Kit Benefits
Lipodin‐Pro™ can be used in various functional studies for cell signaling and apoptotic assays, protein‐protein interaction, protein localization and compartment shuttling. When the protein is conjugated to a fluorescent dye, the functional assay can be carried out in living cells under multiple treatments with a single sample. Principal Lipodin‐Pro ™ advantages:
No need for DNA cloning or nucleic acid transfection
No chemical ligation or crosslinking
Serum compatible, no cytotoxicity and biodegradable
Easier 2‐step protocol with ready‐to‐use reagents
Deliver functionally active protein within hours
Higher delivery efficiency with stable cell lines and primary cells
2. Transfected Proteins
Lipodin‐Pro™ successfully delivered numerous proteins in a wide variety of cells:
B and R‐phycoerythrin
bovine serum albumin
‐galactosidase human active caspase‐3, caspase 8 and caspase 9
immunoglobulins (unconjugated or labeled with FITC, TRITC, AlexaFluor®488 and AlexaFluor®546)
MBP‐fusion protein
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Impurities, contaminants and additives present with the protein of interest affect delivery efficiency. We recommend using a protein sample as pure as possible. We noticed that protein preparations containing high contents of detergents or sodium azide were not compatible with protein transfection, whereas glycerol has no effect.
3. Cell Types Tested
Lipodin‐Pro™ is applicable on numerous cell types. This reagent has been successfully tested on a variety of immortalized cell lines as well as primary cells (Table 1). By submitting end‐user data to our Technical Department, we can update this list and improve our technical support to the scientific community. If a particular cell type is not listed, this does not imply that Lipodin‐Pro™ is not working for that type but that it has not yet been tested.
Table 1: Example of cells successfully tested with Lipodin‐Pro™ reagent.
Cell Line Cell Type Source Efficiency3T6 Embryonic fibroblasts Mouse 50% A549 Non‐small cell lung carcinoma Human 50‐80%B16‐F10 Melanoma Mouse 50% BEAS‐2B Bronchial epithelial cells Human 80% BHK21 Fibroblasts (Kidney) Hamster 80‐90%CHO‐K1 Epithelial‐like (Ovary) Hamster 50‐80%COS‐1, COS‐7 Fibroblasts (Kidney) Green Monkey 50‐70%HaCaT Keratinocytes Human 50‐80%HEK‐293 Transformed Embryonic (Kidney) Human 80‐100%HeLa Cervical Epithelial Carcinoma Human 50‐60%Jurkat T cell leukemia Human 50% L929 Fibrosarcoma Mouse 80‐90%K562 Myelogenous leukemia Human 10‐50%MDCK Epithelial (Kidney) Canine 10‐50%N2A Neuroblastoma Mouse 60‐80%NIH3T3 Fibroblasts Mouse 50‐75%Raw264.7 Monocytes/macrophages Mouse 90% U87 Glioblastoma Human 50% Vero 10A1 Epithelial (Kidney) Monkey 50% Primary cells Neurons Rat 50% Glial cells Rat 50%
4. Delivery of B and R‐Phycoerythrin
NIH3T3 A549
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B‐Phycoerythrin (1 µg, Sigma‐Aldrich) was delivered in the indicated cell lines with 2 µl of Lipodin‐Pro™ reagent. Phycoerythrin/Lipodin‐Pro™ complexes were incubated for 24 hours in 24‐well plates before unfixed cells were observed by fluorescence microscopy.
RAW 264.7 BHK21
BEAS-2B BEAS-2B
3T6 HeLa
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R‐Phycoerythrin (1 µg, Molecular Probes) was delivered in the indicated cell lines with 2 µl of Lipodin‐Pro™ reagent. Phycoerythrin/Lipodin‐Pro™ complexes were incubated for 24 hours in 24‐well plates before unfixed cells were observed by fluorescence microscopy.
5. Delivery of Beta‐Galactosidase
HeLa CHO-K1
A549 A549
Vero Raw 264.7
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‐Galactosidase (1 µg) was delivered in various cells with 2 µl of Lipodin‐Pro™ reagent. ‐Galactosidase/Lipodin‐Pro™ complexes were incubated for 24 hours in 24‐well plates before fixed cells were stained for beta‐galactosidase activity.
6. Delivery of BSA‐TRITC
Tetramethylrhodamine‐labeled BSA (BSA‐TRITC, 2 µg) was delivered in various cells with 3 µl of Lipodin‐Pro™
reagent. BSA‐TRITC/Lipodin‐Pro™ complexes were incubated for 24 hours with cells in 24‐well plates before unfixed cells were observed with a fluorescent microscope.
BEAS-2B NIH3T3
Jurkat BHK21
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7. Delivery of MBP Fusion Protein
The MBP‐fusion protein (10 µg) was delivered with 25 µl of Lipodin‐Pro™ reagent in HEK 293 cells. After an 8‐hour incubation, cells were fixed and immunostained with an anti‐MBP antibody. Then, the cells were observed by fluorescence microscopy. After a 10‐hour incubation, cells were lysed for protein detection by western‐blot.
8. Delivery of Immunoglobulins
A control antibody labeled with
AlexaFluor488 (0.5 µg) was mixed with 2 µl of Lipodin‐Pro™ reagent and incubated for 24 hours with BHK‐21 cells in 24‐well plates. Then, cells were fixed with 2% pFA and observed by fluorescence microscopy.
A Nuclear Pore Complex Protein antibody
labeled with AlexaFluor488 (0.5 µg) was mixed with 2 µl of Lipodin‐Pro™ reagent and incubated for 24 hours with BEAS‐2B cells in a 24‐well plate. Then, cells were fixed with 2% pFA and observed by fluorescence microscopy.
MBP‐Protein alone MBP‐Protein delivery with the Lipodin‐Pro™
reagent
MBP‐Protein Lipodin‐Pro
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9. Kinetics of R‐Phycoerythrin Delivery in NIH3T3 Cells
R‐Phycoerythrin (1 µg) was delivered in NIH3T3 cells with 2 µl of Lipodin‐Pro™ reagent in 24‐well plates. Cells were collected and fixed with 2% PFA at the indicated time point. The number of fluorescent cells and the mean fluorescence was determined by cytofluorimetry. The mean fluorescence was used to evaluate the amount of R‐Phycoerythrin internalized inside cells.
10. Delivery of Active Caspase‐3 Induces Apoptosis
HeLa cells in a 24‐well plate were treated with 15 ng of active human caspase‐3 (Biovision) mixed with 5 µl Lipodin‐Pro™ reagent. Images were taken 6 h after treatment (top row). The bottom image is a control of cells treated with Lipodin‐Pro™ reagent alone. Identical results were obtained with NIH‐3T3 cells.
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An apoptosis assay was performed to quantify apoptotic cells. HeLa cells were treated with 15 ng of active human caspase‐3 mixed to 5 µl of Lipodin‐Pro™ reagent in 24 well plates. After a 7‐hour incubation, cells were stained with both Annexin‐FITC and propidium iodide. Apoptotic and dead cells were counted by cytofluorimetry. A positive control with staurosporine (100 nM) was used to induce apoptosis. Negative controls were cells treated with caspase 3 or Lipodin‐Pro™ alone.
In another set of experiments, HeLa and A549 cells were treated with 15 ng of active human caspase‐3 mixed to 5 µl of Lipodin‐Pro™ reagent in 24 well plates. After a 24‐hour incubation, live cells were counted in each well and
results plotted as percentage relative to non‐treated cells. A positive control with staurosporine (1 M) was used to induce apoptosis. Negative controls were cells treated with caspase 3 or Lipodin‐Pro™ alone.
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Apoptotic Cells % 86 52 11 8
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Stau 1 M Caspase-3 Lipodin-Pro Caspase-3 Non-treated +Lipodin-Pro
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Overall, Lipodin‐Pro™ reagent can efficiently deliver various types of proteins in different living cell lines. The delivery efficiency is cell type and protein dependent with higher yield for acidic proteins. The delivered protein is still active once inside the cell for binding to target proteins and activating cellular pathways.
11. Technical Support
If you need assistance with your experiments using this product, please contact our Technical Support Department: ABBIOTEC 7985 Dunbrook Rd., Ste A San Diego, CA 92126, USA Toll Free: 1 800 854 7453 Telephone: 1 858 586 0500 Fax: 1 858 586 6252 Email: [email protected] Web: www.abbiotec.com
12. Product Use Limitations
Abbiotec, LLC agrees to provide its customers with products under the following terms and conditions. By purchasing and receiving Abbiotec products, customers will agree to the following: All Abbiotec products are provided solely for research use only. Abbiotec products have NOT BEEN APPROVED FOR HUMAN USE, directly or indirectly. Customers agree not to administer any Abbiotec products to human subjects in any manner or form or to use Abbiotec products for therapeutic or diagnostic purposes. Abbiotec customers will also agree not to use its products commercially without prior consent and without the presence of a signed agreement between customer and Abbiotec, LLC. Licenses for diagnostic, therapeutic, or commercial resale use of Abbiotec products may be available and can only be obtained by contacting Abbiotec directly. Customers agree to abide and comply by all applicable local and federal laws that govern the use of research products for research use only. This includes the correct use of Abbiotec products as intended and described in the product manual, and the safe handling and disposal of these products according to all applicable governmental regulations. These product use limitations represent the entire terms and understandings between Abbiotec, LLC and its customers. These use limitations supersede all previous communications, representations, understanding and agreements, whether oral or written, by or between Abbiotec, LLC and the customer regarding the use of Abbiotec products. No change, modification, extension, termination or waiver of any of these use limitations shall be valid unless made in writing and signed by a duly authorized representative of Abbiotec. Abbiotec trademarks referred to in this publication: From Biology to Discovery™, Lipodin‐Ab™, Lipodin‐Pro™. “Abbiotec” and the Abbiotec logo are trademarks of Abbiotec, LLC. All content contained in this manual is property of Abbiotec, LLC and may be used only with the expressed written permission of Abbiotec, LLC. All rights reserved. Trademarks of other companies referred to in this publication: AlexaFluor® (Invitrogen, Inc.).