Protein Delivery Reagent
Contents
1. BioPORTER 34 Protein Delivery Reagent Genlantis A division of Gene Therapy Systems Inc Cat Contents Quantity Shipping Shipped on Dry Ice BP502401 BioPORTER Reagent dried 1 tube Storage Store cells at 20 C Stable for 1 year 24 rxns B galactosidase control protein 10 ug 100 ug ml FITC antibody control protein 10 ug 100 ug ml fluorescein labeled goat IgG Related Products BP509604 BioPORTER Reagent dried 4 tubes 96 rxns B galactosidase control protein 10 ug 100 ug ml BioPORTER Reagent QuikEase 24 single use BP502424 BioPORTER Reagent QuikEase 96 single use BP502424 BioPORTER FITC Antibody Control 10 yg ABFITCO1 BioPORTER 6 gal Control 10 ug BGALCP01 FITC antibody control protein 10 ug 100 ug ml fluorescein labeled goat IgG Introduction The BioPORTER Protein Delivery Reagent is an efficient and trusted reagent for intracellular delivery of bioactive molecules such as proteins peptides and antibodies into a broad range of cell types Although there are many effective reagents available to introduce transcriptionally active DNA into viable cells The BioPORTER Reagent was designed specifically for the delivery of functional peptides and proteins into living cells using a unique lipid based carrier system The BioPORTER Reagent is effective easy to use and more economical than both microinjection and electroporation for the delivery of biolog2. least 2 hours Optionally vacuum the BioPORTER Reagent film for 2 more hours Incubate for 4 hours Preparation of BioPORTER Protein Mix Dilute protein of choice in HBS or PBS buffer Concentration depends on the molecules used 50 250 pg ml is suggested Add diluted protein solution directly to BioPORTER dry film and mix by pipetting Use Table 3 in Step A 6 as a guide Incubate at room temperature for 3 5 minutes Vortex BioPORTER protein mix briefly bring volume up with serum free medium to appropriate level see Table 4 Step A 7 Transfer the mixture onto cells Add serum containing medium if cells continue to incubate longer than 4 hours Example Protocols SPONBDAPWNHARPINOAPWNH gt Galactosidase or FITC Ab Delivery in a 24 well Plate 22 mm Cover Slips Seed 0 5 1 x 105cells per well in 24 well plate or on cover slips and let grow overnight Aliquot 2 5 ul of BioPORTER dissolved in 250 ul of solvent into an eppendorf tube Evaporate the solvent in a cell culture hood for at least 2 hours Optionally vacuum the BioPORTER Reagent film for 2 more hours Dilute 1 ug of protein in 10 ul of HBS Ab or PBS 8 Galactosidase Hydrate BioPORTER Reagent dry film with 10 pl of the diluted protein solution and mix by pipetting up and down 3 to 5 times Incubate at room temperature for 5 minutes Vortex BioPORTER protein complex briefly bring up final volume to 250 ul in serum free medium Blot dry coverslips and put in 35 mm
3. or their components for internal research use only as described in this manual in particular research use only excludes and without limitation resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis a division of Gene Therapy Systems Inc GTS separate licenses are available for non research use or applications BioOPORTER Reagent and or its components are not to be used for human diagnostic or included used in any drug intended for human use Care and attention should be exercised in handling the kit components by following appropriate research laboratory practices Purchasers may refuse this license by returning the enclosed materials unused By keeping or using the enclosed materials you agree to be bound by the terms of this license The laws of the State of California shall govern the interpretation and enforcement of the terms of this License Genlantis Telephone 858 457 1919 e 888 428 0558 U S Toll free Fax 858 623 9494 www genlantis com VKM061101 Page 4 of 5 APPENDIX Quick Reference Protocol for Experienced Users General Protocol Preparation of BioPORTER Reagent 1 Dissolve each BioPORTER Reagent tube in 250 pl chloroform or methanol 2 Vortex for 10 seconds at high speed 3 Aliquot the appropriate volume of BioPORTER Reagent to the bottom of eppendorf tubes 4 Evaporate the solvent in a cell culture hood for at
4. the recommended concentrations but it may exhibit some cytotoxicity at higher reagent cells concentration ratios Tissue Culture Dish BioPORTER range pl 96 well 0 25 1 5 24 well 1 25 5 12 well 2 5 7 5 6 well 5 15 60mm dish 15 30 100mm dish 25 45 4 Optimize the volume used to hydrate the BioPORTER Reagent do this after identifying the correct amounts of BioPORTER Reagent and protein to use in Step A 6 To test this parameter fix the protein amount and vary the hydration volume for BioPORTER Reagent see Table 3 in Step A 6 5 Try different protein dilution buffers Tris HBS and PBS can be tested For some molecules the buffer used may be critical for example PBS buffer works well with B galactosidase but not the Tris buffer With dextran sulfate HBS is the best buffer tested 6 Try different buffer pH pH may be critical for some molecules because of their different charge and hydrophobicity varying the pH may improve interaction with the BioPORTER Reagent 7 Optimize cell numbers delivery efficiency may be sensitive to the confluency of the cells in culture 8 Vary incubation times depending on the type of functional assay performed shorter or longer incubation time may influence delivery efficiency LIMITED LICENSE The purchase price paid for the BioPORTER Protein Delivery Reagent hereto BioPORTER Reagent grants end users a non transferable non exclusive license to use the kits and
5. ul of methanol or chloroform into the bottom of each eppendorf tube Evaporate the solvent as described in Step A 3 above Dilute 0 5 2 yg of FITC Ab dextran sulfate or B galactosidase in 10 to 25 ul of HBS or PBS For galactosidase we recommend using PBS The FITC Ab and galactosidase control proteins provided in the kit are ready to use without further manipulation just thaw and mix them well before use Hydrate the BioPORTER Reagent dry film with 10 25 ul of the diluted protein solution Pipette 3 5 times to mix Incubate at room temperature for 3 5 minutes then vortex briefly and gently at low speed for few seconds Bring the final volume of the BioPORTER protein mixture to 250 ul with serum free medium For 24 well plates aspirate the medium wash once with serum free medium optional then transfer the BioPORTER protein mixture directly onto the cells For cover slips blot the cover slip dry and place it in a 35 mm dish Transfer the BioPORTER protein mixture directly onto the cells Incubate cells in a 5 CO2 incubator at 37 C for 4 hours If incubation time is longer than 4 hours add 250 ul 1 volume of 20 serum containing medium directly to each 24 well or 1 to 2 ml of growth medium to the 35 mm dish containing the cover slip After the incubation wash the cells twice with PBS and proceed with the appropriate assay For fluorescent microscopy after washing cells growing on cover slips are mou
6. 5 to 60 ng ul A Preparation of the BioPORTER Reagent NOTE Experimental results suggest that some though not 1 6 Use the diluted protein solution to hydrate the dried 2 To avoid evaporation of solvent immediately pipette the desired volume of BioPORTER into an a said tube see BioPORTER Reagent The amount of protein peptide Table 1 below for suggestions Be sure to dispense the antibody or other molecules to be delivered will depend on BioPORTER solution to the bottom of the tube ne type of experiment cell type assay sensitivity plate size etc See Table 3 below for suggested amounts NOTE The volume of BioPORTER needed varies depending on the experiment cell type assay sensitivity Table 3 Suggested Quantities of Proteins and Hydration Volumes plate size etc We recommend starting with 2 5 ul of Tissue Protein _Protein Protein BioPORTER BioPORTER solution per reaction in a 24 well plate or 10 ul Culture Ab B gal Caspase3 Granzyme Hydration for a 6 well plate We also highly recommended optimizing Plate Type ug ng B ug Volume ul delivery conditions by varying the amount of 96 well 0 1 0 25 0 25 0 5 0 01 0 05 10 protein peptide to be delivered first then varying the ra z ri ge amount of BioPORTER solution Swell 5 10 10 20 032 50 100 Table 1 Suggested BioPORTER Volumes 60mm 10 20 20 40 0 5 3 100 400 Tissue Culture BioPORTER Number of 100mm 25 50 50 100 0 75 4 250 500 Plate Type Volume u
7. Reagent reacts quickly and interacts non covalently with the protein peptide or other molecule creating a protective vehicle for immediate delivery into cells The hydrated mixture is then added onto cells and BioPORTER Protein the BioPORTER protein complexes attach to negatively Complexes charged cell surfaces The BioPORTER Reagent can then fuse directly with the plasma membrane and deliver the captured protein into the cells see 1 in the figure to the right or the BioPORTER protein complexes are endocytosed by the cells and then fuse with the endosome releasing the BioPORTER captured protein into the cytoplasm see 2 in the figure to the right Delivery of molecules with the BioPORTER Reagent is very easy and requires only 4 hours 4 of incubation with the target cells TOO z Fy Endosome Nucleus VKM061101 Genlantis Page 1 of 5 Telephone 858 457 1919 e 888 428 0558 U S Toll free Fax 858 623 9494 www genlantis com METHODS AND PROCEDURES The conditions that follow are starting guidelines only For best performance we recommend optimizing component concentration cell number time of incubation and protein hydration buffers Further optimization guidelines are provided in the Optimization Guideline Section on page 4 Table 2 Protein Concentration Ranges Examples Antibody B gal or dextran sulfate Caspase 3 Granzyme B 50 250 pg ml 0 05 0 3 units yl 165 to 1000 pg l 7
8. an also be at time points earlier than 4 hours Page 3 of 5 Telephone 858 457 1919 e 888 428 0558 U S Toll free Fax 858 623 9494 www genlantis com OPTIMIZATION GUIDELINES It is highly recommended to optimize your conditions in order to get the best BioPORTER Reagent performance The following parameters can be optimized Amount of protein or molecule to be delivered Buffer used to dilute the protein Amount of BioPORTER Reagent Concentration of the protein solution Hydration volume for the BioPORTER Reagent Cell types and cell culture density Time of incubation Optimize one parameter at a time as follows 1 Start_by using a fixed amount of BioPORTER for example use 2 5 ul of BioPORTER Reagent per reaction in a 24 well plate 2 Vary the amount of protein to be delivered Use a standard buffer for example HBS or PBS Depending on the sensitivity of the endpoint assay a greater amount of protein and BioPORTER Reagent may be required 3 If further optimization is required fix the concentration and amount of protein to be delivered and then vary the quantity of the BioPORTER Reagent used see table below The BioPORTER Reagent interacts with your molecules of interest via hydrophobic and electrostatic interactions and because each molecule will have different charge and hydrophobicity the amount of BioPORTER Reagent may need to be changed NOTE the BioPORTER Reagent is not cytotoxic at
9. dish or for 24 well plates Aspirate old medium Transfer BioPORTER protein medium mixture to cells 10 Incubate cells in a 5 CO2 incubator at 37 C for 4 hours 11 Add serum containing medium if incubation time needs to be longer than 4 hours 12 After incubation wash cells and proceed with the appropriate assay NOo PON gt Delivery of Apoptotic Proteins Granzyme B Caspase 3 or Caspase 8 Seed 0 5 x 105 adherent cells per well in a 24 well plate and grow overnight For suspension cells see Step 7 below Pipette 2 5 ul of BioPORTER Reagent dissolved in 250 ul of solvent into an eppendorf tube Evaporate the solvent in a cell culture hood for at least 2 hours Optionally vacuum the BioPORTER Reagent film for 2 more hours Dilute caspase 3 to 330 pg ul 0 1 units ul and granzyme B to 30 ng ul in HBS Dilute B gal to 0 1 yg pl in PBS for negative control Add 10 ul of the diluted protein solution to BioPORTER Reagent dry film and mix by pipetting up and down 3 to 5 times Incubate at room temperature for 3 5 minutes Vortex BioPORTER protein complexes briefly then for adherent cells bring final volume to 200 ul with serum free medium aspirate media then transfer BioPORTER protein mix directly onto cells for suspension cells count and pellet cells resuspend in serum free medium to 0 5 x 10 cells ml Pipette 200 ul of cell suspension into BioPORTER protein mix then transfer to 24 well plate 8 Incubate cells in a 5 CO2 i
10. ically active proteins into living cells The specific formulation of the BioPORTER Reagent can deliver various molecules over a broad range of cell types in serum free conditions and within 3 to 4 hours of incubation Various molecules such as fluorescent antibody high and low molecular weight dextran sulfate phycoerythrin BSA B galactosidase caspase 3 caspase 8 and granzyme B have been successfully delivered with the BioPORTER Reagent into the cytoplasm of a variety of different adherent and suspension cellst Furthermore apoptotic proteins such as granzyme B caspase 3 or caspase 8 drove cells into apoptosis after delivery with the BioPORTER Reagent confirming that BioPORTER delivers functional proteins into cells Now you can make your macromolecules directly available for a variety of studies like intercellular signaling cell cycle regulation control of apoptosis study of oncogenesis and transcription regulation to name a few t For a list of citations and cell types successfully used with the BioPORTER Reagent visit our web site at www genlantis com Summary of the BioPORTER Reagent Protein Delivery Mechanism The dried BioPORTER Reagent formulation is first dissolved in a solvent and aliquoted into small eppendorf tubes according to the type of assays conducted see Methods and Procedures After complete drying the BioPORTER Reagent is formulated with a solution of the protein or peptide to be delivered The BioPORTER
11. ing the following buffers to the size of plate dish and transfection volume Pipette HBS 10 mM HEPES 150 mM NaCl pH 7 0 the cell suspension into the tube of BioPORTER protein BES EO mM Na phosphate 150 mM NaCl pH 7 4 mixtures and then transfer it to your well or dish Tris Buffer 10 mM Tris 150 mM NaCl pH 7 0 NOTE The presence of serum in the first hours of 5 The final concentration of your proteins or molecules of incubation inhibits protein delivery ate ee aa Bara AN ae 10 Incubate for 3 4 hours at 37 C If longer incubation time is i i 0 a few concentration ranges that yielded good results pi PO poling SUEN PINS coe PERUN VKM061101 Genlantis Page 2 of 5 Dissolve each tube containing the dry film of BioPORTER Reagent with 250 ul of methanol or chloroform Vortex for 10 20 seconds at top speed before each use all highly positively charged molecules are not efficiently delivered into cells because they interact poorly with the BioPORTER Reagent see Page 4 for suggestions Telephone 858 457 1919 e 888 428 0558 U S Toll free Fax 858 623 9494 www genlantis com EXAMPLE PROTOCOLS C D VKM061101 Delivery of a Fluorescent Antibody Galactosidase or Dextran Sulfate High and low Molecular Weights for 24 well Plates or 22 mm Cover slips Seed 0 5 to 1 x 105 cells per well in a 24 well plate or on a cover slip and let grow overnight Pipette 2 5 ul of BoPORTER solution dissolved in 250
12. l reactions kit 96 well 1 240 7 Add serum free medium to the BioPORTER protein 24 well 2 5 96 complex to bring the final delivery volume up to the a 7 a amounts recommended in Table 4 below we 60mm dish 20 12 Table 4 Suggested BioPORTER Solution Volumes 100mm dish 35 7 Tissue Culture Number of Total Delivery Plate Type Cells Volume 3 Leave the eppendorf tubes containing the BioOPORTER 96 well 1 2 x 104 100 pl solution under a laminar flow hood to evaporate the solvent 24 well 0 5 1 x 105 250 ul for at least 2 hours at room temperature For larger 12 well 1 2 x 105 500 pl volumes evaporate for at least 3 4 hours Complete 6 well his 1 ml evaporation of the solvent is essential e oa Pi eat NOTE Alternatively an inert gas e g argon nitrogen can ee P A PE aH SaR 8 Aspirate medium from the cells to be tested wash once gas flow to the BloPORTER solution at the bottom of the vii SELU ER Pe Aini epuatial y ANGE ten MASIE ane tube The dried BioPORTER film may also be vacuumed Mal aewary mbvcniocels for 1 2 additional hours to remove all traces of solvent 9 For adherent cells directly add the BioPORTER protein complexes in serum free medium onto the washed cells B Preparation of the BioPORTER Protein Complexes For suspension cells first count the cells centrifuge them l at 1200 rpm for 5 minutes and then resuspend them in Die the protein peptide or other molecule in one of serum free medium Adjust their concentration accord
13. ncubator at 37 C for 4 hours add 1 2 ml medium 10 serum directly to wells incubate overnight 9 The next day proceed with the apoptosis assay Troubleshooting Guide Problem Possible Causes Recommended Solutions Low delivery Solubilization e Make sure you use chloroform or methanol to solubilize BioPORTER Reagent vortex vigorously efficiency Drying e Use sufficient time to air or vacuum dry BioPORTER Reagent Do not splash with inert gas Amount of BioPORTER Reagent e Vary the amount of BioPORTER Reagent as recommended in the optimization protocol Protein peptide concentration e Titrate the concentration and the hydration volume of the BioPORTER Reagent Hydration buffers e Change the protein dilution buffer and or the pH to improve the delivery Mixing BioPORTER and protein e Allow mixtures to form for at least 3 minutes Mix well by pipetting do not vortex at this step Charge of molecules to be delivered e Highly positively charged molecules are difficult to deliver modify the hydration buffer or pH Cell Density e Use cells that are 50 60 confluent Wrong medium used e Make sure to use serum free medium during the first hours of delivery Improper storage e BioPORTER Reagent is stable but exposure to elevated temperatures may cause degradation Time of incubation e Incubate BioPORTER protein complexes with cells for at least 3 4 hours Type of cell line used e Use positive cont
14. nted directly onto a hanging drop slide with PBS Observe living or fixed cells under a microscope For galactosidase assay X Gal staining For best results we recommend using the Genlantis X Gal staining Kit cat A10300K the protocol for this kit is available at www genlantis com Delivery of a Fluorescent Antibody 8 Galactosidase or Dextran Sulfate for a 6 Well Plate or 35 mm Dish Seed 2 x 105 cells in 6 well plate and let grow overnight Pipette 10 ul of BioPORTER Reagent dissolved in chloroform or methanol into the bottom of each eppendorf tube Evaporate solvent as in Section C 2 above Genlantis Dilute 5 10 ug of protein in 50 100 yl of appropriate buffer as in Step C 3 above Hydrate the BioPORTER Reagent dry film with 50 100 ul of the diluted protein solution Pipette up and down 3 to 5 times Incubate at room temperature for 5 minutes then vortex briefly and gently at low to medium speed Bring the final volume of the BioPORTER protein mixture to 1000 ul with serum free medium Aspirate the medium from the cells to be tested wash one time with serum free medium optional and then transfer the BioPORTER protein mixture directly onto the cells Incubate cells in a 5 CO2 incubator at 37 C for 4 hours If incubation time needs to be longer than 4 hours add 1000 ul 1 volume of 20 serum containing medium directly to the well Delivery of Granzyme B and Caspase 3 Into Jurkat or Ki Ras 267 1 Cell
15. rols along with cell lines tested successfully visit www genlantis com for a list Aggregation Amount of BioPORTER Reagent e Too much BioPORTER Reagent could cause aggregation try using a lower amount Evaporation of stock solution e Excessive evaporation of the dissolved Bio PORTER Reagent will change its concentration Titrate down or use lower amounts Storage e BioPORTER protein complexes should be freshly prepared if not aggregation may occur Cytotoxicity Excess BioPORTER reagent e Decrease the amount of BioPORTER Reagent used Molecules delivered are toxic e Use the appropriate control reactions cells alone BioPORTER Reagent alone control protein alone along with your test protein and check the purity of the molecule of interest to be delivered Unhealthy cells Check cells for contamination or use a new batch of cells Cells are too confluent or cell density is too low Check the culture medium pH kind used last time changed etc Check materials used for proper function culture plates incubator temperatures etc
16. s for 24 Well Plates For adherent cells such as Ki Ras 267 B1 prostate cancer seed 0 5 x 105 in 24 well and let cells grow overnight For Jurkat cells see Step 6 below Pipette 2 5 wl of BioPORTER Reagent dissolved in chloroform or methanol into the bottom of each eppendorf tube Evaporate the solvent as in Step C 2 above Dilute caspase 3 to 330 660 pg ul or granzyme B to 15 45 ng ul in HBS buffer buffer recipes are in Step B 4 above Hydrate the BioPORTER reagent dry film with 10 ul of the diluted protein solution Pipette up and down 3 5 times Incubate at room temperature for 3 5 minutes vortex briefly and gently at low to medium speed for few seconds For adherent cells such as Ki Ras 267 1 bring the final volume of the BioPORTER protein mixture to 200 ul with serum free medium Aspirate the medium wash once with serum free medium optional and then transfer the BioPORTER protein mixture directly onto the cells For suspension cells such as Jurkat count and pellet the cells resuspend them in serum free medium to 0 5 x 108 cells ml Add 200 ul of the cell suspension to the BioPORTER protein mixture and then transfer the mix to a 24 well plate Incubate cells in a 5 COz incubator at 37 C for 4 hours Add 1ml of serum containing medium directly to the well and incubate overnight The next day proceed with the apoptosis assay using any commercially available annexin V propidium iodine labeling kit This assay c
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