amaxa Peptide Transfection Control 2-3 Product specifications 2 Storage and stability 3 Product use limitations 3 Intended use 3

Transcription

1 Contents amaxa Peptide Transfection Control 2-3 Product specifications 2 Storage and stability 3 Product use limitations 3 Intended use 3 Background Information 4 Cell culture 4 Supporting Data 5-8 Equipment required before starting 7 Peptide preparation 7 Peptide quality 7 Quality control 8 Peptide concentration 8 Transfection procedure 8 Post-transfection steps 8 Troubleshooting Guide 9 Related Products 1 License Statement 11 Scientific Support 12 Page 1 Contents

2 amaxa Peptide Transfection Control Peptide Transfection Control molecular weights and characteristics. [k] Cat. No. VBT-11 DMA-18 Kit Contents: 5 μg Peptide Transfection Control (lyophilized) Peptide Transfection Control Manual Total Peptide molecular weight Fluorophore molecular weight 14.2 Da 146 Da 354 Da Peptide modifications C-terminus amide Product specifications Mean hydrophilicity N-terminus FAM -.3 (25% hydrophilic residues) 1.2 Normalized fluorescense Ex Em Isoelectric point Net charge at ph HO O O Wavelength (nm) Figure 1. Excitation & emission spectra of the amaxa Peptide Transfection Control. Spectra of the amaxa Peptide Transfection Control Excitation (Ex. max) Emmission (Emm. max) Peptide purity (HPLC) Peptide length 482 nm 522 nm > 95% 8 amino acids Figure 2. Peptide Transfection Control fluorophore (FAM) structure. COOH COOH Storage and stability The expiration date is printed on the reagent box. For maximum shelf-life, the amaxa Peptide Transfection Control should be stored lyophilized at -8 C in an air tight and light-proof container. Product use limitations The amaxa Peptide Transfection Control is intended for research and investigational use by professionals only. The amaxa Peptide Transfection Control is not approved for any clinical application and must not, under any circumstances, be used for testing or treatment in humans, or as a therapeutic or diagnostic tool, or as an accessory or complement to such a tool. Page 2 Peptide Transfection Control Page 3 Peptide Transfection Control

3 Intended use The amaxa Peptide Transfection Control is a fluorescently labeled octapeptide intended for introduction into the cytoplasm and nuclei of primary cells and cell lines, when used in conjunction with Nucleofector and 96-well Shuttle Technology. The amaxa Peptide Transfection Control is ideally suited for use with both the Nucleofector and the 96-well Shuttle Systems. Up-to-date recommendations and transfection tips for your specific cell types are available at the amaxa cell database: Alternatively, please contact amaxa Scientific Support. Supporting Data Figure 3: Delivery of Peptide Transfection Control into different cell lines. amaxa Peptide Transfection Control offers: An easy standardization method for functional peptide analyses Reproducible Nucleofection for validation and control experiments A control reference for troubleshooting peptide transfection efficiency amaxa support for Nucleofection of peptides A: Jurkat cells (1 6 ) were transfected with 15 μm Peptide Transfection Control. Cells were analyzed by light and fluorescence microscopy directly after Nucleofection. 25 [k] Background Information Cell culture For commercially available cell lines and primary cells we recommend following the instructions of the supplier (e.g. ATCC ) regarding culture medium and supplement as well as passage and seeding conditions. Optimal transfection results will be obtained when cell culture conditions are standardized. Please refer to the amaxa Cell Database for recommendations regarding your specific cell type: Mean fluorescence (RLU) HEK293 SH-SY5Y Jurkat B: Bar histogram showing transfection efficiency as analyzed by flow cytometry (intensity of fluorescence) in HEK293, SH-SY5Y and Jurkat cells transfected with amaxa Peptide Transfection Control. 1. Control (no peptide, no Nucleofection ); 2. Peptide Transfection Control without Nucleofection ; 3. Peptide Transfection Control with Nucleofection ). Page 4 Backround Information Page 5 Supporting Data

4 Luciferase Activity (%) Peptide Transfection Control with Jurkat cells peptide + peptide Nucleofection ctrl TNF-alpha stimulation Figure 4. Specific inhibition of luciferase gene expression in a stable NF-kB-luciferase HEK-293 cell line. NF-kB Reporter HEK-293 cells (Panomics ) stably expressing a reporter luciferase gene under the control of the transcription factor NF-kB exert luciferase activity via the NF-kB pathway when stimulated with TNF-alpha 1. No TNF-alpha stimulation; 2. TNF-alpha stimulation (5 ng/ml); 3. Incubation with a peptide that interferes with this signaling pathway (25 μm) results only in minor inhibition of luciferase activity, probably due to low permeability of the cells to peptide; 4. Nucleofection of the inhibitory peptide (25 μm) results in a significant inhibition of luciferase activity by delivering peptide directly into the cell Control 1 μm 1 μm 2 μm 2 μm 3 μm 3 μm Nucleofection Nucleofection Nucleofection Figure 5. Linear increase of X-mean with increasing extracellular concentration of Peptide Transfection Control. Jurkat cells were exposed to Peptide Transfection Control at the illustrated concentration with, and without, Nucleofection. Fluorescence X-mean (Relative Light Units) was measured by flow cytometry for each sample. Mean values generated from 3 replicates within a single experiment are shown. Equipment required before starting Molecular biology grade dimethyl sulfoxide (DMSO, water-free) 1-2% (w/v) BSA in 1x Phosphate Buffered Saline (PBS) Sterile water Calibrated pipettes (1-2 μl and 2-1 μl) & sterile, disposable tips 1-2 sterile, screw-top tubes for peptide aliquots -8 C freezer Peptide preparation Recommendation To yield a 1 mm stock solution, dissolve the amaxa Peptide Transfection Control in 4 μl of 2% DMSO. Ensure that the amaxa Peptide Transfection Control is completely dissolved. Please consider the influence of DMSO on the cells you will transfect and select a concentration appropriate for use in the final transfection volume (Nucleofector 1 μl; 96-well Shuttle System 2 μl). DMSO concentration should not exceed 3% in the final volume. Once the amaxa Peptide Transfection Control is dissolved, the solution should be dispensed to tubes in quantities suitable for use in a single experiment and stored in a light-proof container at -8 C until required. N.B. Avoid repeated freeze-thawing of the amaxa Peptide Transfection Control. Peptide quality Peptide purity may influence both the viability of transfected cells and also the functionality of any transfected peptide. amaxa recommends the use of highly pure (> 95%) peptides from a reputable supplier, such as INTAVIS AG. Page 6 Supporting Data Page 7 Supporting Data

5 Quality control Each batch of Peptide Transfection Control is functionally tested in conjunction with Nucleofection and compared to results obtained using a reference batch. Peptide concentration amaxa recommends an initial working concentration of 15-3 μm (e.g μl of the 1 mm stock solution per 1 μl Nucleofection volume) when first validating peptide uptake in your primary cells or cell lines. Validation of uptake for each new cell type used, and each time the cell culture conditions are changed in any way sufficient to alter the cellular phenotype, is also recommended. Transfection procedure Please refer to the amaxa Optimized Protocol appropriate for your cell type. Troubleshooting Guide Maximize both the validity and reproducibility of your results by ensuring that test peptides and control peptides are transfected in the recommended Nucleofector Solution and by harmonizing the methods used for both test peptides & Peptide Transfection Control. Issue High background fluorescence Low fluorescence of the Peptide Transfection Control or test peptides in transfected cells Probable cause Unspecific binding of peptides on cell surface Peptides are subjected to unspecific breakdown and export mechanisms Recommended action Include washing steps: Centrifuge cells and resuspend in PBS/BSA to remove extracellular bound peptides. Repeat washing steps as necessary. To extend peptide lifetime and retention time, slow down cellular metabolism by using chilled reagents and chilling cells on ice after Nucleofection and during washing steps. Post-transfection steps In order to reduce background fluorescence of cells due to surface binding of Peptide Transfection Control, wash the cells using PBS (1-2% BSA). Low fluorescence of the Peptide Transfection Control or test peptides in transfected cells Peptides are subjected to unspecific breakdown and export mechanisms If it is not possible to keep your samples at low temperature during the procedure you might choose an earlier time point of analysis to assure the highest intra cellular concentration of peptide. Cells should be pelleted by centrifugation as recommended in the relevant amaxa Optimized Protocol (8-1 x g) and washed by gentle aspiration in 1 x PBS containing 1-2% BSA. Low cell viability after washing steps Cell damage by washing procedure Reduce centrifugation speed during your washing steps and ensure that cells are washed by gentle aspiration. Low cell viability with your test peptide Toxic effects of your test peptide Examine the toxicity of your peptide (e.g. homology to known cytotoxic peptides). Low cell viability with your test peptide Toxic effects of your test peptide. Reduce the concentration of your test peptide to decrease toxic effects. Page 8 Supporting Data Page 9 Troubleshooting Guide

6 [k] Related Products Please refer to the amaxa cell database for kits recommended for the cell type you are using and the accompanying Optimized Protocols. License Statement Purchaser shall not manufacture, copy, reproduce, transmit, distribute, sell, lease, transfer or sublicense the amaxa Peptide Transfection Control, unless the Purchaser has negotiated a license for commercial use with amaxa AG. Cat. No. Expression vectors VDF-111 VDF-112 VDF-113 VDF-121 VDF-122 VDF-123 VDF-131 VDF-132 VDF-133 Product pmaxfp -Green-C Vector pmaxfp -Green-N Vector pmaxfp -Green-PRL Vector pmaxfp -Yellow-C Vector pmaxfp -Yellow-N Vector pmaxfp -Yellow-PRL Vector pmaxfp -Red-C Vector pmaxfp -Red-N Vector pmaxfp -Red-PRL Vector Copyright amaxa 28. amaxa, Nucleofection, Nucleofector, 96-well Shuttle, maxgfp and maxfp are either registered trademarks or trademarks of amaxa AG in Germany and/or the U.S. and/or other countries. All other product and company names mentioned herein are the trademarks of their respective owners. Antibiotics VZA-112 Plasmocin (5 mg) VZA-121 Primocin VZA-11 Normocin (5 mg) VZA-12 Normocin (1 g) All rights reserved. Page 1 Related Products Page 11 License Statement

7 Scientific Support amaxa s scientific support team is composed of scientists with many years of gene transfer experience at post-graduate level. They are dedicated to providing you with comprehensive and accurate answers to your questions via phone, fax or and will respond in the quickest possible time. As they are continuously in contact with amaxa s research team and many different users of transfection products, amaxa s staff is always an excellent source of information for up-to-date news on the most recent transfection developments. USA phone (888) fax (888) scientific-support.us@amaxa.com Europe/World phone +49 () fax +49 () scientific-support@amaxa.com Page 12 Scientific Support