DeliverX™ Peptide Transfection Kit
Contents
1. Results Hydrophillic Peptide Loading Viability Sequence Length Residues 96 MW Charge Index Index4 GIKNNLKDCGLD 12 50 1794 38 0 3 5 GIKNNLKECGLT 12 45 1793 38 1 2 5 CGLHDNLKQLMLQ 13 38 2019 38 1 1 4 GNGIKCLFNDKL 12 42 1826 38 1 5 4 CGGRMAPPRRDAMPSDA 17 35 2293 38 1 5 5 SIRKALNILGYPDYD 15 40 2243 38 0 3 5 MPKKKPTPIQLNP 13 38 1997 38 3 5 5 KGRKPRDLELPLSPS 15 53 2197 38 2 3 5 GSFLVRES 8 50 1398 38 0 2 5 RFARKGALRQKN 12 58 1950 38 5 3 5 RFAAKGALRQKN 12 50 1865 38 5 5 5 PVKRRLFG 8 38 1477 38 3 4 5 PVKRRLDL 8 50 1501 38 2 4 5 PVKRRLFL 8 38 1533 38 3 2 5 DAAREGFLDTAVVAHRAGAR 20 30 2587 38 1 5 5 CTMNRRGAIKQAK 13 46 1982 38 4 4 4 CTMNARGAIKQAK 13 38 1897 38 3 2 5 CPRKRQGAVRRRV 13 54 2087 38 6 5 4 CPRKAQGAVRRRV 13 48 2002 38 5 3 5 CTRKRQRAMRRRV 13 62 2223 38 7 5 5 CTRKAQRAMRRV 13 54 2138 38 6 5 5 DeliverX Peptide Transfection Kit User Manual Page 19 Appendix I Delivery of Polar Peptides Hydrophillic Peptide Loading Viability Sequence Length Residues MWb Charge Index Index CSIYRRGARRWRK 13 5496 2213 38 6 5 5 CSIYARGARRWRK 13 4696 2127 38 5 5 5 GRAGNQYL 8 38 1383 38 1 3 4 GGLPPFRAG 9 11 1376 38 1 1 5 a Peptides consist of FITC and aminohexanoic acid linker at the N terminus and amide modification at the C terminus b Molecular weight of peptides linker and FITC c Loading index quali2. 2 5 5 Aboutthe Kits 5 omni Be Ala lekt paileith Debbie x M ded 5 About the Manual ssssssslseelll eee 5 DeliverX Peptide Transfection Kit Contents and Storage 000s 6 Contents and Storage of DeliverX Kit llle 6 Storage Recommendations llle ees 6 Safety Warnings and Precautions ssssoseessesessesss esse eee 6 Required and Recommended Materials Not Provided 200005 6 Materials Required But Not Provided llli rss rr rr era 6 Materials Recommended But Not Provided lille ses ses ena 6 Assay Workflow and Recommended Guidelines Controls and Optimization 7 Assay Workflow serias tiidrit e iil eed a ete otc deem n 7 Transfection Guidelines 00000 cece eee 7 Recommended Controls 00 0c cece ete eee 7 Optimizing Transfections ssoossssseesssass ss sr rss rss rh 8 Recommendations for Peptide Design and Preparation ss ssssssssssssss soo 8 Peptide Designin nen ette CUR Gate ue t a 8 Preparing Peptide Cargos isssseseeseeeee e 9 Preparing the Cells the Day Before Transfection illie 10 Adherent Cell Types smosssssssrsss saras ssk eee 10 Preparing the Peptide Peptide Transfection Reagent Complex 10 About Preparing the Complex 0000 00 e eee ee eens 10 Procedure 2 1 Bee ete ERA ates Lande Death dubbed teda foetu eed aches 11 Transfecting Adherent Cells
3. b Carefully wash the cells twice with pre warmed PBS c Add Hank s Balanced Salt solution For extensive viewing use Hank s solution plus 296 serum d Examine and image the cells using a fluorescent microscope Note Use TAMRA filter Ex Em 542 568 for TAMRA Peptide Control or Pre Formed Control 5 Add complete growth media At this step the final concentration of peptide in the sample is 1 3 uM without dilution of the working stock DeliverX peptide complex Per Well of a Complete Growth Media pL 6 well plate 900 12 well plate 600 24 well plate 450 96 well plate 90 6 Incubate under normal cell culture conditions typically at 37 C and 5 CO for 24 hours or desired time intervals For example the induction of cell cycle arrest using CDK2 inhibiting peptide requires analysis after 24 48 hours of incubation after the peptide transfection complex addition However the induction of ERK1 2 phosphorylation by G gamma protein inhibiting peptide requires analysis within 15 minutes of incubation after the peptide transfection complex addition Transfecting Cells in Suspension Reverse Transfection About Transfecting Cells in Suspension Assay Preparation Procedure Page 14 This procedure describes the transfection of adherent cells after trypsinization and while they are in suspension Transfect adherent cells in suspension when you want to perform high throughput screening of many peptides on a single cell type Gr
4. of 500 nM and 600 nM respectively for example TAMRA or Rhodamine Minimize the usage of fluorophores that require 365 488 nm excitation wavelength for example FITC Dapi or Coumarin DeliverX Peptide Transfection Kit User Manual Appendix I Delivery of Polar Peptides Appendix I Delivery of Polar Peptides About this This section briefly describes the experimental details that were used to perform Appendix transfections using the DeliverX Peptide Transfection Kit and summarizes the results in tabular format Experimental HeLa cells were seeded at 7 500 cells well into a 1 5 glass bottom 96 well plate Conditions The next day 1 3 uM FITC labeled peptide complex was added to each well followed by the addition of serum free media Cells were incubated for 2 hours and then complete growth media was added Cells were incubated for an additional 2 hours before they were gently washed twice with PBS Complete growth media was added and cells were viewed under the microscope to determine transfection efficiency loading and viability index Similar experiments were performed on NIH3T3 cells In general transfection efficiency was greater than 7096 when the loading index see Scale of Loading Index on page 20 for a definition of this metric was greater than 3 Position and intensity of hydrophobic polar and neutral regions were analyzed using peptide property calculation software provided at www innovagen com
5. sssossoossesessssss rss ss rss eee 13 Assay Preparation 0 0 00 hn 13 Procedures xd sov aet dea eet da ete RAND TG gett sadder 13 Transfecting Cells in Suspension Reverse Transfection s sss ss s sss os so 14 About Transfecting Cells in Suspension illie 14 Assay Preparation 22d so cxtat dun E Bd Eee ga Ee 14 Procedure ico eh si IUE ER IN OUI RI PU 14 Troubleshooting vx der tale cee te oot e gead woe PEMEX a ura quad 17 Problems and Recommended Actions llli lle ss eae 17 Appendix I Delivery of Polar Peptides llle 19 About this AppendDc sui ioa td Deda Bek pee domi A Pa wag 19 Experimental Conditions 0 000000 cee ses 19 FesullS ier cfe rr UU made tee Ou de UE ee ER edi ea Rd 19 Scale of Loading Index liiis eres 20 ATP Viability ASSay 5 zie ettet ut e eot Suas 20 Functional ASSay o5 ee excede v ebet d ber ebbe vedete diet d 21 Contacting Pariomlics 1 zc necat ast BIG oe afa Zac Sra tr Ze xg ac ARA Parce 22 DeliverX Peptide Transfection Kit User Manual Page iii Table of Contents Page iv Technical Help For Additional Services DeliverX Peptide Transfection Kit User Manual DeliverX Peptide Transfection Kit and Manual Overview DeliverX Peptide Transfection Kit and Manual Overview About the Kits Our DeliverX Peptide Transfection Kit is suitable for transfection of most cell types DeliverX Peptide Transfection Kits contain the rea
6. sufficient for the transfection of 1 well of a 6 12 24 or 96 well plate To prepare DeliverX peptide peptide transfection reagent complex Step Action 1 Thaw peptides and DeliverX Peptide Transfection Reagent and store on ice 2 Prepare peptide working stocks a Dilute the peptides to 160 uM with 1X PBS b Sonicate in the water bath sonicator at maximum output and power for 5 minutes If peptides have aggregates or precipitates centrifuge as described in Preparing Peptide Cargos on page 9 3 Dilute the 160 uM peptide working stocks with Buffer 1 in 1 5 mL tubes as described in the table below 160 uM Peptide Per Well of a Working Stocks pL Buffer 1 uL 6 well plate 15 60 12 well plate 10 40 24 well plate 7 5 30 96 well plate 1 5 6 4 Prepare the DeliverX Peptide Transfection Reagent a Sonicate the DeliverX Peptide Transfection Reagent at maximum output and continuous power for 3 5 minutes to achieve a homogenous solution b Prepare dilutions in 1 5 mL tubes as described in the table below IMPORTANT Sonication of DeliverX Peptide Transfection Reagent is critical for achieving good complex formation Ensure that the tubes are submerged in the water during sonication DeliverX Peptide Per Well of a Transfection Reagent pL Buffer 2 uL 6 well plate 60 15 12 well plate 40 10 24 well plate 30 7 5 96 well plate 6 1 5
7. Carefully remove media from the wells and wash once with PBS Per Well of a 1X PBS uL 6 well plate 500 12 well plate 300 24 well plate 150 96 well plate 100 Add the working peptide peptide transfection reagent complex Working DeliverX Per Well of a Peptide Peptide Complex pL 6 well plate 300 12 well plate 200 24 well plate 150 96 well plate 30 IMPORTANT Manually rock do not swirl the plate to evenly distribute the complex over the well surface Add complete growth media and mix by gentle rocking Note Peptide concentration if undiluted is 2 6 uM Per Well of a Complete Growth Media pL 6 well plate 600 12 well plate 400 24 well plate 300 96 well plate 60 DeliverX Peptide Transfection Kit User Manual Page 13 Transfecting Cells in Suspension Reverse Transfection To transfect adherent cells continued Step Action 4 Incubate under normal cell culture conditions typically at 37 C and 596 CO for 4 hours Proceed to the next step Note For maximal loading we recommend 4 hours Optional For some peptides and cell types delivery efficiency and loading capacity increase by using serum free media instead of serum containing media For cells transfected with fluorescent labeled peptide a Incubate under normal cell culture conditions for 0 5 4 hours
8. DeliverX Peptide Transfection Kit User Manual amp Panomics Panomics Inc DeliverX Peptide Transfection Kit User Manual Copyright Copyright 2006 Panomics Inc All rights reserved Trademarks DeliverX is a trademark of Panomics Inc QuantiGene is a registered trademark exclusively licensed to Panomics Inc All other trademarks belong to their respective owners Citing DeliverX in Publications When describing a procedure for publication using this product we would appreciate it if you would refer to it as the DeliverX M Peptide Transfection Kit If a paper cites a DeliverX product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Licenses DeliverX Peptide transfection reagents are manufactured and distributed by Panomics under license from CNRS France Disclaimer Panomics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Contents DeliverX Peptide Transfection Kit and Manual Overview
9. MRA Peptide Control or Pre Formed Control Add complete growth media At this step the final concentration of peptide in the sample is 1 3 uM without dilution of the working stock DeliverX peptide complex Per Well of a Complete Growth Media pL 6 well plate 900 12 well plate 600 24 well plate 450 96 well plate 90 10 Incubate under normal cell culture conditions typically at 37 C and 5 CO for 24 hours or desired time intervals For example the induction of cell cycle arrest using CDK2 inhibiting peptide requires the analysis after 24 48 hours of incubation after the peptide transfection complex addition However the induction of ERK1 2 phosphorylation by G gamma protein inhibiting peptide requires the analysis within 15 minutes of incubation after the peptide transfection complex addition DeliverX Peptide Transfection Kit User Manual Troubleshooting Troubleshooting Problems and To troubleshoot transfections using DeliverX Peptide Transfection kits Possible Cause Recommended Action Peptide is unable to form transfection complex due to one or more of the following Not soluble Does not contain hydrophobic domains Does not contain positive or negative charged domains Less than 10 amino acids in length Follow the guidelines in Recommendations for Peptide Design and Preparation on page 8 Non functional peptide Redesign pep
10. and more than 2596 of Dissolve in 2596 acetic acid then dilute with the amino acids are charged 1X PBS Note Use the smallest amount of acetic acid possible to minimize the impact on cell physiology DeliverX Peptide Transfection Kit User Manual Page 9 Preparing the Cells the Day Before Transfection If the peptides are Then Positively or negatively charged but fewer Dissolve in 10096 DMSO Add DMSO than 2596 of the amino acids are charged drop wise with constant agitation Sonicate to ensure complete dissolution Dilute with 1X PBS Note Complex formation is not affected by up to 396 DMSO Note Use the smallest amount of DMSO possible to minimize the impact on cell physiology Remove aggregates by sonication for 5 minutes followed by centrifugation at 200 000 x g for 30 minutes We recommend the AirFuge Ultracentrifuge from Beckman Coulter Microcentrifuges that generate 14 000 x g can also be used but might not produce optimal results Store dissolved peptide at 80 C Preparing the Cells the Day Before Transfection Adherent Cell For most adherent cell types the optimal confluency for transfection is 60 9096 The Types following table provides guidelines for seeding differently sized culture vessels to obtain 60 9096 confluence after 24 hours of growth IMPORTANT Use cells from passages 4 20 cell type specific Optimal cell density is very important for obtaining the best resul
11. ansfection Kit User Manual Page 17 Troubleshooting Page 18 To troubleshoot transfections using DeliverX Peptide Transfection kits continued delivery pattern of fluorescent Observation Possible Cause Recommended Action Peptide Peptides are not completely Dissolve in desired solvent See aggregates soluble Required and Recommended Materials Not Provided on page 6 for more information Ultra centrifuge to remove aggregates Non diffuse Peptides stick to plate Avoid using collagen matrigel fibronectin or poly lysine coated plates fluorescent labeled peptide peptide Switch to another plate type Peptide cargo contains Sonicate and centrifuge peptide cargo insoluble aggregate before mixing with DeliverX peptide Unable to Sensitivity of the Grow cells on 170 um thick 111 5 glass observe microscope setup is poor substrate such as a standard glass cover slip or glass bottom microwell Use a microscope with high Numerical Aperture lens and fluorescent filter setup See Required and Recommended Materials Not Provided on page 6 High background due to peptides or DeliverX Peptide complex binding non specifically to cell culture plate or substrate Switch to a different culture vessel type or format Avoid using collagen matrigel fibronectin or poly lysine coated plates Signal masked by auto fluorescent cells Use fluorophores that excite and emit at the range
12. c After diluting vortex briefly and sonicate again at maximum output and continuous power for 3 5 minutes DeliverX Peptide Transfection Kit User Manual Page 11 Preparing the Peptide Peptide Transfection Reagent Complex Page 12 To prepare DeliverX peptide peptide transfection reagent complex continued Step Action 5 Form concentrated peptide cargo peptide transfection reagent complex a Mix the peptide cargo Buffer 1 solution from Step 3 with the Peptide Transfection Reagent Buffer 2 solution from Step 4 b Incubate tubes at RT for 30 minutes c Prepare complex dilution buffer by mixing equal volumes of Buffer 1 and Buffer 2 d Add an equal volume of complex dilution buffer to the peptide peptide transfection complex The concentration of peptide is 8 uM at this point Without further dilution final peptide concentration after addition of growth media will be 1 3 uM To make lower concentrations dilute using complex dilution buffer Note After incubation the concentrated peptide peptide transfection reagent complex can be stored at 20 C for up to 6 months The concentrated complex is stable for up to 10 freeze thaw cycles DeliverX Peptide Transfection Kit User Manual Transfecting Adherent Cells Transfecting Adherent Cells Assay Preparation Pre warm both serum free and complete growth media to 37 C Procedure To transfect adherent cells Step Action 1
13. ee an adequate response The maximum peptide concentration that DeliverX Peptide Reagent can deliver is 1 3 uM final concentration A good starting range for peptide concentration is 0 5 1 3 uM Recommendation for Optimizing Cell Density and Peptide Concentration We recommend you perform a two parameter matrix experiment that includes 3 different concentrations for example 0 5 1 and 1 3 uM final concentration and 3 cell densities for example 60 75 and 9096 confluency and select the cell density and peptide concentration that yields the best cell viability and biological response Recommendations for Peptide Design and Preparation Peptide Design Page 8 In general peptides with the following characteristics are efficiently delivered into most cell types However even petides with the following characteristics might not be efficiently delivered into the cells DeliverX Peptide Transfection Kit is well suited for peptides with the following characteristics Loading capacity amount of peptide that enters the cell increases with increasing number of these characteristics Longer peptides 210 amino acids are preferred DeliverX Peptide Transfection Kit User Manual Recommendations for Peptide Design and Preparation More than one hydrophobic region consisting of a minimum of 3 continuous or alternating aromatic and aliphatic amino acids Amino acids with the highest hydrophobicity are best For example xxxHHHxxx or xH
14. en P N 70013 032 Hank s balanced salt solution Invitrogen P N 14025 Ultrasonic cleaner bath sonicator with 30 40 kHz sonication Panomics Inc P N DX0400 or equivalent Item Source TAMRA labeled Peptide Control Panomics Inc P N DX1100 Pre Formed Peptide Transfection Complex Control Panomics Inc P N DX1101 Airfuge ultracentrifuge Beckman Coulter Airfuge Fluorescent microscope Olympus Model IX71 20X objective lens Numerical aperture 0 75 Olympus P N 1 U2B825 TAMRA filter cube Ex Em 542 568 nm Omega Optical P N XF 32 311 5 coverglass base optical bottom black 96 well microplate NUNC P N 164588 Ammonium bicarbonate MLS Acetic acid MLS DMSO MLS DeliverX Peptide Transfection Kit User Manual Assay Workflow and Recommended Guidelines Controls and Optimization Assay Workflow and Recommended Guidelines Controls and Optimization Assay Workflow Time Required 1 Prepare Adherent Cells Variable typically 16 24 hr a Plate cells before transfection 2 Prepare Transfection Complex 30 min a Prepare Peptide Cargo Buffer 1 b Prepare Peptide Transfection Reagent Buffer 2 c Combine Peptide Cargo Buffer 1 and peptide Transfection Reagent Buffer 2 d Incubate complex at RT for 30 min 3 Transfect Cells 24 72 hr a Add complete media or serum free media incubate 2 4 hr at 37 C 5 CO b Add complete med
15. eptide E am Results of a similar experiment performed by Goubaeva et al using myristoylated Gg peptide Contacting Panomics Technical Help For Additional Services Page 22 For technical questions contact our technical support group by telephone at 877 726 6642 or by email at techsupport panomics com or visit our website www panomics com for an updated list of FAQs and product support literature For information about Panomics products or for ordering information contact your Regional Sales Manager or visit our website at www panomics com DeliverX Peptide Transfection Kit User Manual
16. gent Transfection Reagent Negative Control at 48 hr Cdk2 peptide at 48 hr HeLa cells grown in 96 well microplate were transfected with Cdk2 inhibiting peptide using DeliverX Peptide Transfection Reagent and incubated for 48 hours before the brightfield images were obtained Gg Binding Peptide Induces Phosphorylation of Erk 1 2 in Rat 2 Fibroblast Tyrosine Kinase Linked Receptor G protein coupled Receptor Stimulation Inhibition Stimulation or Inhibition Cellular Responses Flow chart illustrating the Known G protein activation of ERK 1 2 pathways Gg peptide SIRKALNILGYPDYD binds to G Beta Gamma subunit and induces the dissociation of G alpha subunit The associated G Beta Gamma subunit activates the MAPK pathway resulting in increased phosphorylation of ERK 1 23 1 Image courtesy of The Science Creative Quarterly www scq ubc ca Jane Wang illustrator 2 J Biol Chem 2008 Vol 278 19634 19641 3 Image courtesy of www vascularweb org DeliverX Peptide Transfection Kit User Manual Page 21 Contacting Panomics Negative PMA Gp peptide peptide Induced DelX DelX DelX DelX P Erk1 2 GAPDH 0 0 LL G peptide was transfected into Rat2 cells using DeliverX Peptide Transfection Reagent Rat 2 cells were lysed 15 minutes after transfection and western blotting was performed to measure phosphorylation of ERK 1 2 GAPDH is shown as a control for loading Negative peptide Gg p
17. gents required to efficiently transfect peptides into most cell types with minimal cell damage and good reproducibility when following the optimization guidelines provided in this User Manual The DeliverX Peptide Transfection Kit is designed to efficiently deliver a wide range of hydrophobic and polar peptides that interfere with the function of targeted cellular proteins We have successfully transfected 40 FITC labeled peptides into the following cell lines with no visible effects on cellular morphology and with cell viabilities over 7096 Cell Line 9o Efficiency of Transfection NIH3T3 gt 70 HeLa gt 70 3T3L1 gt 70 C2C12 gt 70 Differentiated C2C12 gt 30 Human astrocytes CCF STTG1 gt 70 MEF gt 70 DeliverX Transfection Mechanism DeliverX transfection reagents are based on the novel delivery technology called MPG This technology was developed at Centre de Recherches en Biochimie Macromol culaire CNRS in Montpelier France in the laboratory of Dr F Heitz and Dr G Divita MPG technology uses virus derived amphipathic peptides that directly interact with peptide cargos to form non covalent nanoparticles 150 200 nm capable of diffusing through the plasma membrane and releasing their contents inside the cell The mechanism of entry is receptor independent involves MPG lipid interactions and avoids the endocytic pathway thereby preventing endosomal or lysosomal degradation of car
18. gos About the Manual The manual contains a description of the kit contents and guidelines recommendations and procedures for performing transfections using the DeliverX Peptide Kit DeliverX Peptide Transfection Kit User Manual Page 5 DeliverX Peptide Transfection Kit Contents and Storage DeliverX Peptide Transfection Kit Contents and Storage Contents and Storage of DeliverX Kit Storage Recommendations Safety Warnings and Precautions DeliverX Peptide Transfection Kit Contents Component Storage DeliverX Peptide Transfection Reagent 20 C DeliverX Peptide Buffer 1 RT DeliverX Peptide Buffer 2 RT Divide transfection reagent into aliquots to minimize freeze thaw cycles Store components at recommended temperatures Product shelf life is 6 months from date of shipment if stored properly CAUTION All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice For research use only Do not use internally or externally in humans or animals Required and Recommended Materials Not Provided Materials Required But Not Provided Materials Recommended But Not Provided Page 6 Item Source Cell culture reagents and equipment Major laboratory suppliers MLS Phosphate buffered saline pH 7 2 PBS Invitrog
19. ia incubate 24 72 hr at 37 C 5 CO 4 Quantify Delivery by Fluorescence Microscopy or Functional Assay Variable depending on assay Transfection Use healthy cells in mid log phase of growth not overgrown Guidelines 4 Use cells between 4 20 passages passage number is cell type specific Do not use antibiotics during transfection process You can use antibiotics to maintain the cell line Sonicate the transfection reagent as stated in the procedure to ensure proper formation of the DeliverX peptide transfection reagent complexes Note Once the DeliverX peptide transfection reagent complex has been formed properly it can be diluted to obtain varying peptide concentrations Perform all procedures in a laminar flow hood using proper tissue culture techniques Recommended Proficiency Controls Controls For new users or when working with a new cell type we recommend the use of the following proficiency controls DeliverX TAMRA labeled pre formed peptide to determine if DeliverX Peptide Reagent is compatible with the cell line TAMR labeled peptide to assess the complex formation and transfection procedure Fluorescent labeled peptide cargos to determine the transfection efficiency However note that conjugating fluorescent dye may impact the delivery of peptide into cells DeliverX Peptide Transfection Kit User Manual Page 7 Recommendations for Peptide Design and Preparation Optimizing Transfectio
20. low Working DeliverX Per Well of a Peptide Peptide Complex pL 6 well plate 300 12 well plate 200 24 well plate 150 96 well plate 30 6 IMPORTANT Manually rock do not swirl the plate to evenly distribute the complex over the well surface 7 Immediately add complete growth media as indicated in the table below and mix by gently rocking Per Well of a Complete Growth Media pL 6 well plate 600 12 well plate 400 24 well plate 300 96 well plate 60 DeliverX Peptide Transfection Kit User Manual Page 15 Transfecting Cells in Suspension Reverse Transfection Page 16 To transfect cells in suspension continued Step Action 8 Incubate under normal cell culture conditions typically at 37 C and 596 CO for 4 hours Proceed to the next step Note For maximal loading we recommend 4 hours Optional For some peptides and cell types delivery efficiency and loading capacity increase by using serum free media instead of serum containing media For cells transfected with fluorescent labeled peptide a Incubate under normal cell culture conditions for 0 5 4 hours b Carefully wash the cells twice with pre warmed PBS c Add Hank s Balanced Salt solution For extensive viewing use Hank s solution plus 296 serum d Examine and image the cells using a fluorescent microscope Note Use TAMRA filter Ex Em 542 568 for TA
21. ns Routine Controls These controls assess the transfection and peptide knockdown efficiency and we recommend they be used routinely Positive control peptide to ensure that the assay is working in a reproducible manner This is a validated high potency peptide whose functional response is known Cells only to serve as a mock transfection designed to monitor for any non transfection related phenomenon during the experiment This control contains the transfection buffers but does not include either the transfection reagent or peptide cargos Note DeliverX Peptide transfection reagent alone should not be used as a negative control because of its high affinity for the cell membrane uncomplexed reagent might be cytotoxic Because cell types can differ significantly with respect to their capacity to be transfected we recommend that you optimize the protocol empirically The most important parameters for optimization are cell density and peptide concentration Cell Density Cells that are too dense or too sparse may not take up an optimal amount of the complexes resulting in minimal effect on functional phenotypes or elevated levels of cytotoxicity Peptide Concentration A peptide s capacity for eliciting a phenotypic response is influenced in part by the peptide design stability and nature of its interaction with the target If too much peptide is used during transfection you may see toxicity If too little is used you may not s
22. ow adherent cells so that they are 70 9096 confluent on the day of transfection Pre warm both serum free and complete growth media to 37 C To transfect cells in suspension Step Action 1 Trypsinize and pellet cells by centrifugation DeliverX Peptide Transfection Kit User Manual Transfecting Cells in Suspension Reverse Transfection To transfect cells in suspension continued Step Action 2 Gently resuspend cells in PBS using a wide bore pipet and pellet again 3 Gently aspirate the media avoiding cell loss and resuspend the cells in PBS to the concentration as indicated in the table below For a Resuspend to cells mL 6 well plate 3 0 9 0 x 106 12 well plate 1 0 4 0 x 106 24 well plate 1 0 3 0 x 106 96 well plate 0 5 1 0 x 106 4 Gently pipet the cell suspension into the plate as indicated in the table below Per Well of a Add uL of the Cell Suspension 6 well plate 100 12 well plate 50 24 well plate 25 96 well plate 10 Note For experiments in which you will be transfecting fluorescent labeled peptide such as TAMRA labeled peptide control and viewing the cells under a microscope we recommend that you grow the cells on a 41 5 coverslip or glass bottom microplate for optimal detection of fluorescent signal 5 Add the working DeliverX peptide peptide transfection reagent complex as indicated in the table be
23. tative measurement using intensity of FITC signal in cells to determine the relative amount of peptide that enters the cell see images below d Viability index qualitative measurement using cell density and morphology to estimate viability For more information see ATP viability assay below Scale of Loading Scale 1 Scale 2 Scale 3 Index HeLa cells transfection with TAMRA labeled peptide control ATP Viability Assay Cell Viability ATP assay Hela Cell Viability ATP assay NIH 3T3 E 140 F 140 a a hr 8 120 100 100 Ohr z5 4hr 23 9 zs Ww hr S y 80 Fe a So O8hr 88 gt 9 60 23 O8hr a24hr 2 6096 8 40 3 x o24hr S 209 m 48hr AM 40 E 20 m 48hr 0 E 20 Ss 1 3 uM 0 43 uM 0 14 uM 0 uM 0 1 3 uM 0 43 uM 0 14 uM 0 uM Peptide Concentration Peptide Concentration Peptide Transfection Complex was added to cells and ATP levels were measured at 0 4 8 24 and 48 hours Page 20 DeliverX Peptide Transfection Kit User Manual Appendix I Delivery of Polar Peptides Functional Assay Inhibition of Cell Proliferation by Cdk2 Peptide in HeLa Cells S Replication Mite rp G1 Gap 1 cell grows cell prepares to divide c itosis Cells that cell division cease division Left Cartoon of the cell cycle Right HeLa cells transfected with FITC labeled Cdk2 peptide o Aq e e 3 DeliverX Peptide DeliverX Peptide Transfection Rea
24. tide for increased binding affinity to the target protein or test additional peptides Cell density not optimal Evaluate cell densities outside of the recommended 60 9096 confluence at the time of transfection Transfection time not adequate Evaluate transfection times greater than 4 hours Cell have a tendency to grow in groups or clumps Do not tap flask during trypsinization Allow the cells to detach themselves After trypsinizing the cells pipet up and down several times to release the cells Seed cells at the desired density Visually check cell density using a light microscope Cell response changes after repeated passages Thaw fresh cells for subsequent experiments Avoid using cells at early or late passages Serum interferes with delivery Reduce the amount of serum or use serum free media as described in the procedure Recommended Observation Actions Expected phenotype is not observed High toxicity Cell density too low Evaluate higher cell densities at the time of transfection Be gentle when removing medium or PBS during washes Cells become more sensitive to reagents after repeated passages Thaw fresh cells for subsequent experiments Avoid using cells at early or late passages Too much working transfection complex added to the cells Follow the recommended guidelines stated in the manual for optimizing transfection DeliverX Peptide Tr
25. ts Note For experiments in which you will be transfecting fluorescently labeled peptides such as TAMRA labeled peptide control and viewing the cells under a microscope we recommend that you grow the cells on a 1 5 coverslip or glass bottom microplate for optimal detection of fluorescent signal If you are using a Then seed cells at a density of In a volume of 6 well plate 150 300 x 103 cells well 2 mL well 12 well plate 50 200 x 10 cells well 1 mL well 24 well plate 25 75 x 10 cells well 500 uL well 96 well plate 5 10 x 108 cells well 100 uL well Note These numbers are approximate because the exact number of cells required depends on cell type size and growth rate Preparing the Peptide Peptide Transfection Reagent Complex About Preparing Sonication of the DeliverX Peptide Transfection Reagents per the procedure below is the Complex critical for proper formation of the complex used for transfection Once the complex has been properly formed it is amenable to dilution for evaluating a range of peptide delivery concentrations This novel peptide based delivery system requires no Page 10 DeliverX Peptide Transfection Kit User Manual Preparing the Peptide Peptide Transfection Reagent Complex optimization of the peptide to transfection reagent ratio and enables high efficiency peptide transfection typically with 1 3 uM or less Procedure The following procedure prepares transfection complexes
26. xHxHxxx where H hydrophobic amino acid See below for more information More than one charged region at pH 7 2 consisting of 3 continuous or alternating negatively and positively charged amino acids For example XXxCCOxxx or xCxCxCxxx where C positively or negatively charged amino acid at pH 7 2 Positively charged peptides are preferred over negatively charged peptides Atleast 3 or more Arg or Lys amino acids at the C or N terminus Completely soluble in 1X PBS pH 7 2 Amino Acid Property Phe Trp Tyr Highly hydrophobic aromatic Lle Leu Gly Val Ala Moderately hydrophobic aliphatic Arg Lys His Positively charged at neutral pH Asp Glu Gln Asn Negatively charged at neutral pH Pro Met Cys Gly Neutrally charged at neutral pH For more information on peptide properties visit www innovagen com For more information on amino acid properties visit web indstate edu thcme mwking amino acids html www mcb ucdavis edu courses bis102 AAProp html Preparing Peptide Peptides must be soluble and contain no aggregates Use the following guidelines for Cargos dissolving peptides If the peptides are Then Negatively charged and more than 2596 of Dissolve in 0 1 M ammonium bicarbonate the amino acids are charged then dilute with 1X PBS Note Use the smallest amount of ammonium bicarbonate possible to minimize the impact on cell physiology Positively charged
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