ALVEOLE Révision 3. PRIMO Protocol for Protein Patterning. CONFIDENTIEL page 1/10

Transcription

1 PRIMO Protocol for Protein Patterning CONFIDENTIEL page 1/10

2 TABLE OF CONTENTS 1 Purpose of this Protocol Protocol writing symbols Property and Copyright Warnings Laser risk Chemical risk Equipment needed Laboratory equipment Consumables and reagents Personnal protective equipment Drawing the pattern Protein patterning experiment Surface treatment Protein patterning APPENDIX... 9 CONFIDENTIAL Page 2 / 10

3 1 Purpose of this Protocol This Protocol has no contractual value and in no case will Alvéole be held responsible on the bases of information contained in this manual. This protocol details all the necessary steps to make a pattern of proteins with the PRIMO system. Thus, after careful reading, the operator will be able to: Prepare the glass surfaces for protein patterning Draw and photopattern the pattern for his choice with a fluorescent or non-fluorescent protein The company Alvéole edits this protocol as is, without warranty, explicit or implicit, including, but not limited to, implied warranties of merchantability and/or fitness for a particular purpose. Despite every effort made to propose a protocol that is as accurate as possible, this protocol may contain technical inaccuracies and/or typographical errors. In no case will Alvéole be held responsible for any loss of profit, loss of business, loss of data, business interruption, or any indirect, special, accidental or consequential damages. In the event of damage due to a default (an imperfection) or an error in the protocol, Alvéole commits to send to the user as soon as possible a paper or electronic document containing any corrections made to this manual. This protocol is regularly updated. The most recent version of this protocol is available on simple request to Alvéole. However, should important modifications be made to the protocol, Alvéole commits to send to the user as soon as possible a paper or electronic copy of the updated protocol. It is stated that this does not include the update of the equipment and/or the software in your possession. The product owner is required to keep this protocol throughout the entire duration of use. Any request for information or modification concerning this protocol should be addressed to: Alvéole, 68 Boulevard de Port-Royal, PARIS, FRANCE. 1.1 Protocol writing symbols This symbol means: LASER RISK The instructions preceded by this symbol may cause, for the user and their immediate surroundings, bodily harm to the eyes or the skin, if not respected or carried out. This symbol means: CAUTION The instructions preceded by this symbol may cause bodily harm for the user, or tamper the PRIMO system or the whole set-up, if not respected or carried out. This symbol means: INFORMATION Complementary information with no effect on the implementation of this protocol. CONFIDENTIAL Page 3 / 10

4 1.2 Property and Copyright All manuals and documentation of any kind are the property of Alvéole and are protected by copyright, all rights reserved. Your right to copy this documentation is limited to the rights of the legal copy. These manuals may not be distributed, translated or reproduced, in whole or in part, in any manner and in any form whatsoever without the prior written permission of Alvéole. Thus, reproduction, adaptation or translation of this manual without prior written permission is prohibited within the limits set by the laws governing copyrights. Copyright 01/ /2017 Alvéole All rights reserved. 2 Warnings 2.1 Laser risk The UV Laser of the Optical case is a class 3B laser. Laser radiation: do not look directly into the laser beam with the naked eye or with an optical instrument. 2.2 Chemical risk Danger The PLPP should be handled as a toxic substance. It has an acute toxicity by ingestion and skin contact and can cause eye irritation and skin irritation. Wear appropriate personal protective equipment during use: gloves and goggles Reference document: Product safety data sheet: ALVEOLE PRIMO SDS PLPP EN.pdf CONFIDENTIAL Page 4 / 10

5 3 Equipment needed 3.1 Laboratory equipment To perform a protein patterning experiment the laboratory shall have or have access to the following equipment: - Plasma cleaner and dedicated pump - Laminar flow hood or, failing that, a Sorbonne - Inverted microscope - Motorized XY-stage - Camera - PRIMO - Control software LEONARDO - Computer 3.2 Consumables and reagents The following consumables and reagents are necessary to perform a protein patterning of experiment: Ultra-clean glass coverslip 22x22 mm or 24x60 mm Cut PDMS spacer: o 9 wells of 3x3 mm o 4 wells of 4 mm in diameter o Any design Glass Petri dish Plastic Petri dish of 35 mm in diameter Parafilm Narrowpointed tweezers Micropipettes and adequate tips PLL-PEG 1x solution in HEPES 10 mm ph = 7,4 PLPP 1x solution Protein of interest in solution, C = µg/ml PBS 1x solution ph= 7,4 The equipment described here is the standard configuration. You can replace the ultra-clean coverslip by the substrate of your choice. CONFIDENTIAL Page 5 / 10

6 3.3 Personal protective equipment To conduct a protein patterning experiment the use of adequate personal protective equipment is required: Safety glasses adapted to UV radiation (see section 2.1 Laser risk) Safety glasses adapted for handling chemical (see section 2.2 Chemical risk) Gloves 4 Drawing the pattern Draw the desired pattern using ImageJ, Inkscape, Illustrator, Matlab or any other drawing software; the final image must have the following properties in order to be loaded in the LEONARDO software: Size: 1824 x 1140 pixels Format: tiff or bitmap Type: 8-bits You should draw the image directly in pixels so that it can be interpreted by the DMD. Use the Table 1 to calculate in X and Y directions, the pixel size drawing to obtain the desired size in µm on the sample. Please consider: Lpix image: the size in pixel to draw on the image Léch: the size in µm desired on the sample MAG: the actual magnification of the lens used Tcaméra: the size in µm of the camera pixel The GNx and GNy ratios are calculated by the LEONARDO software during calibration. Table 1: Correspondence charter for pattern (pix)/ sample (µm) Orientation X Léch MAG Lpiximage = 2 GNx Tcaméra Orientation Y Léch MAG Lpiximage = GNy Tcaméra CONFIDENTIAL Page 6 / 10

7 To completely cover the DMD, you should upload images of 1824x1140 pixels in the software. 5 Protein patterning experiment 5.1 Surface treatment The first step of the protocol aims to cover the surface to pattern with a anti-fooling polymer that prevents proteins from adhering on the surface. If possible, all instructions written below in red will be done under a laminar flow hood (clean environment). It is essential that the well surface does not dry during the whole duration of the experiment. Do not touch the bottom of the well with the pipette tip, during the rinsing process, in order to avoid leaving marks on the surface Realize the following steps: Prepare the spacer of PDMS by removing areas corresponding to the well Place the coverslip in a glass Petri dish and close it Introduce the Petri dish in the plasma cleaner then remove the top of the Petri dish Close the plasma reactor: plasma air 3 to 5 minutes Open the plasma and put the top of Petri dish back on before getting it out Place the spacer of PDMS on the coverslip Put 5-10 µl of PLL-PEG 1x in each well (for a square well of 3x3 mm) Put the coverslip in a Petri dish of 35 mm and close it with parafilm Incubate for 1h at room temperature Rinse each well 4 to 5 times with 20 µl of PBS 1x Leave 5-10 µl of PBS 1x in each well CONFIDENTIAL Page 7 / 10

8 5.2 Protein patterning To pattern ONE protein on the surface of a well, proceed as described below: Start the photopatterning setup Start the LEONARDO software Follow the calibration procedure Place the coverslip on the microscope and adjust the focus (use the PFS) Choose a well and aim for the bottom right corner of this well Download the pattern in Leonardo Remove the solution from the well but leave 1µL so that it will not dry out Add 5 µl of PLPP 1x Insolate for about s per pattern using the laser full power (5V) Wear adequate gloves and goggles when working with PLPP (see section 2.2 Chemical risk) When the laser is on, during calibration and UV insolation steps, do not look directly into the beam getting out of the microscope s objective. Wear protective goggles adapted to UV ray (375 nm) (see section 2.1 Laser risk) Rinse the wells 4-5 times with 20 µl of PBS 1x Remove the solution from the well but leave 1µL so that it will not dry out Add 5 µl of protein in solution Incubate for approximatively 5 minutes at room temperature Rinse the well 4-5 times with 20 µl of PBS 1x and change tips every time Leave 5-10 µl of PBS 1x in the well Observe in epifluorescence if needed It is essential that the well do not dry out during the whole duration of the experiment. Regularly add PBS 1x in non-used wells. The protocol described here enables you to pattern one protein. For multiprotein photopatterning, you just have to repeat all the steps, starting with section 5.2, successively adapting the insolation time and concentration of the protein you want to pattern. CONFIDENTIAL Page 8 / 10

9 6 APPENDIX Appendix 1: Experimental sequence for protein patterning CONFIDENTIAL Page 9 / 10

10 Appendix 1: Experimental sequence for protein patterning CONFIDENTIAL Page 10 / 10