MELANIETM version 9
The 2-D Gel Image Analysis Software Package
Platforms: Windows® 11
Important
Please take the time to familiarize yourself with the program by reading the documentation. You will find that the investment in time will help you to quickly become a proficient user of Melanie.
Contents
1	Software modules
1.1	Melanie Classic
1.2	Melanie DIGE
1.3	Melanie Coverage
2	Minimum System Requirements
3	Software installation
3.1	Software updates
4	Software protection - Licensing
4.1	Find the Physical Address
4.2	Install the license server
4.3	Place a license file
4.4	Start the license server
4.5	Start using the license
5	Documentation
6	Data folders
7	Tutorials
7.1	Non-DIGE Tutorial
7.2	DIGE Tutorial
7.3	Coverage Tutorial
8	Uninstall Instructions

1	Software modules
Depending on the product(s) you purchased, this installation of Melanie will give access to one or several of the following software modules. 
1.1	Melanie Classic
This module offers all the functionality needed for differential expression analysis of conventional 2DE gels.
1.2	Melanie DIGE
This module includes all the features of the Classic module, plus specific functionality for differential expression analysis of 2D-DIGE experiments and other multiplexed technologies without internal standard. This lets you fully exploit the advantages offered by multiplexing, in particular the high statistical confidence and reliability resulting from the use of an internal standard.
1.3	Melanie Coverage
This module includes all the features needed to analyze HCP antibody coverage experiments. Both conventional 2D PAGE/Western Blot experiments and 2D-DIGE or 2D-DIBE experiments can be analyzed.
2	Minimum System Requirements
To install and run this software, the following minimum specifications are required:
•	Microsoft Windows® 11 operating system.
•	Administrative privileges to install Melanie, the license server, and the license.
•	Minimum 8 GB of RAM. Increased memory enhances the performance when many and/or large images are analyzed.
•	Minimum 24-bit color resolution and 1024 x 768 screen resolution.
•	Browser to view the software documentation and access databases on the web. Recent versions of Microsoft® Edge, Google Chrome™ or Mozilla™ Firefox™ are recommended.
•	Mouse with scroll function.
3	Software installation
You can install Melanie by registering via this form. Right after registration, you will receive an email with the link to the installation file. Please check your Spam/Junk folder if you don't see it in your incoming email. Make sure to download the installer immediately because the link only remains active for 24 hours. Once you saved the setup file to your computer, you can install whenever you like by double clicking the file icon.
The Melanie installer creates a default directory on your hard disk called Program Files\SIB\Melanie 9, where the program files are placed. To save the installation directory in a different folder, browse and open the folder before continuing the installation.
3.1	Software updates
The software alerts you when a new version of Melanie is available. While it doesn't automatically update, it displays a message with the new version number and directs you to the website to download the latest installer.
If you download and install this newest Melanie release with the aim to update your current version, first make sure that the maintenance period of your license hasn't expired and the license still supports this release. Contact us if you would like to renew your maintenance and benefit from the latest changes and fixes in the software.
Then uninstall the previous release (see Section 8 Uninstall Instructions) and install the new one. Do not remove any files or folders manually. That way, all your data, preferences and license files remain unchanged. Once the software update is complete, you can immediately continue working on your projects.
4	Software protection - Licensing
Upon installation, Melanie is fully functional when analyzing the tutorial images (see Section 7), but it does not allow alignment and detection on other gels. To analyze your own images, you must purchase and install an electronic license, or request a trial license. The availability of the license file is verified each time the software is launched.
There are two types of Licenses:
•	Node-locked license: This license is appropriate for non-networked system environments or for users who require constant availability of Melanie. It is locked to a specific computer, on which the license file must be placed. Melanie will only be able to run in fully functional mode on that computer.
•	Floating license: This license is useful when many users, but not all at the same time, need access to Melanie. With a floating license, Melanie can run on all computers networked to a license server, on which the floating license file must be placed. The number of computers that can simultaneously work with Melanie depends on the license, and is administered via the license server. This server can either be installed on a computer running Melanie or on any other network computer (recommended: install on a network computer that is continually running).
Read on to learn how to obtain and place a license file.
4.1	Find the Physical Address
In order for SIB to create a license file, the Host ID – more particularly the Physical Address of your computer (also called MAC address or Ethernet address) – is needed. This address identifies the computer and is used by the licensing system.
If purchasing a node-locked license, send the Physical Address of the computer where Melanie is to be installed. If purchasing a floating license, send the Physical Address of the computer where the license server is to be installed.
To find the Physical Address:
OPTION 1
1.	On the computer where the license file is to be placed, find the Command Prompt app using the Windows search box.
2.	In the Command Prompt window, enter ipconfig /all. There must be a space between ipconfig and /all.
3.	The response should contain several parameters for the network. Search for an adapter with a name that contains “Ethernet”. Then look for the Physical Address. It is formatted like 30-B0-34-E2-6E-A9. This is the recommended Host ID for the license. While you can use the Physical Address of one of the other adapters, you should avoid virtual adapters or devices that are deactivated in low power mode.
4.	Send the Physical Address to melanie@support.sib.swiss so that your license can be created. Keep in mind that the Physical Address must be correctly recorded for the license file to work.
OPTION 2
1.	On the computer where the license file is to be placed, find the System Information app using the Windows search box.
2.	In the System Summary, click Components > Network > Adapter.
3.	In the list of adapters, search for one with a name that contains “Ethernet”. Then look for the MAC Address. It is formatted like 30:B0:34:E2:6E:A9. This is the recommended Host ID for the license. While you can use the MAC Address of one of the other adapters, you should avoid virtual adapters or devices that are deactivated in low power mode.
4.	Send the MAC Address to melanie@support.sib.swiss so that your license can be created. Keep in mind that the MAC Address must be correctly recorded for the license file to work.
4.2	Install the license server
This section only pertains to floating licenses. If you have a node-locked license, then skip to the next section.
To install the SIB License Server:
NOTE: Administrative privileges are required to install and run the license server, and place the license file. If any question about Windows Firewall appears during the procedure, answer Yes in order for the license server to work properly. If you encounter any issues to start the license service using LMTOOLS (see below), make sure to add lmgrd.exe to the firewall exceptions of your antivirus, and to enable the TCP/IP ports 27000 to 27009 on the server computer, as these are used by the licensing components.
1.	Download and run the installation file http://www.2d-gel-analysis.com/licenseserver/Setup_SIBLicenseServer.msi on the computer where the license server is installed.
2.	In the installation window, click Next.
3.	Accept the default installation path and start the installation. By changing the default installation path, the file paths in LMTOOLS will need to be updated accordingly.
4.	If Windows User Account Control asks if you want to allow the program to make changes to your computer, answer Yes.
5.	LMTOOLS automatically opens once the installation is successfully completed.
6.	Click the Config Services tab and ensure that the following options are set:
•	Service Name: SIB
•	Path to the lmgrd.exe file: C:\Program Files\SIB\SIB License Server\lmgrd.exe
•	Path to the license file: C:\Program Files\SIB\SIB License Server\Licenses\
•	Path to the debug log file: C:\ProgramData\SIB\lmgrd.log
•	Start Server at Power Up: ticked
•	Use Services: ticked
7.	Click Save Service, even if no changes were made.
8.	Leave LMTOOLS open and proceed to the next section.
4.3	Place a license file
You will receive the license file (with a .lic extension) from SIB once you have sent the Physical Address/MAC Address of your computer.
•	For a node-locked license, the file must be placed in the Melanie installation folder (C:\Program Files\SIB\Melanie 9 by default).
•	For a floating license, the file must be placed in the Licenses subfolder of the License server installation folder (C:\Program Files\SIB\SIB License Server\Licenses by default).
4.4	Start the license server
This section only pertains to floating licenses. If you have a node-locked license, then skip to the next section.
To start the SIB License Server:
1.	Select the Start/Stop/Reread tab in LMTOOLS.
2.	Click Start Server.
3.	Close LMTOOLS.
4.5	Start using the license
Node Locked license
Simply start Melanie on the computer where the software and license file are installed. If the software starts in Demo mode:
1.	Click the Import license button.
2.	Check Specify the License File and click Next.
3.	Browse to select the node-locked license file (normally in the Melanie installation directory) and click Open.
4.	Terminate the process by clicking Next, then Finish.
Floating license
1.	Start Melanie on each computer where the software is installed. Click the Import license button in the window that appears.
2.	Check Specify the License Server System and click Next.
3.	Enter the name of the computer where the license server is installed. Enter “localhost” if the license server is running on the same computer as Melanie. Click Next, then Finish.
5	Documentation
Complete and regularly updated documentation (including User Guide, various other guides, FAQs and blog articles) is available online on the website https://2d-gel-analysis.com/documentation/.
Two versions of the User Guide (in HTML format) can be accessed directly from within the software:
•	The online version (https://2d-gel-analysis.com/documentation/) points to the up-to-date documentation of the latest software release.
•	The offline version – automatically accessed when there is no connection to the Internet – points to the locally installed documentation (C:\Program Files\SIB\Melanie 9\Help\user-guide or C:\Program Files\SIB\Melanie 9\Help\user-guide-coverage) of the software release effectively installed by the customer.
6	Data folders
By default, new analysis projects are created in your Local folder, which Melanie establishes within the user's Documents folder and cannot be altered. However, you have the option to designate a Shared folder, typically on a network, to centralize projects for smoother collaboration with colleagues. Once the latter established, you can decide to save new projects either in the Local or the Shared folder.
Please consult https://2d-gel-analysis.com/user-guide-coverage/options-c/ or https://2d-gel-analysis.com/user-guide/options/ for recommendations about choosing an appropriate Shared folder, how to set the folder and move existing projects to it.
Avoid moving or renaming the Local or Shared folders, or manually copying or moving projects from one of these folders to another. Always use the software's built-in functions to duplicate, move, or delete projects to ensure data integrity.
7	Tutorials
Melanie comes installed with a number of tutorial images that you can use to evaluate the software. These images can be found in the folder Tutorials in the software installation directory.
7.1	Non-DIGE Tutorial
The 12 sample gels used in this tutorial come from an experiment that aims to study protein expression changes between four conditions. Cells were grown on two different substrates (substrate A and substrate B) and underwent one of two treatments (treatment 1 and treatment 2). Three biological replicates have been run for each condition.
Condition	Treatment	Substrate
A_T1	Treatment 1	Substrate A
A_T2	Treatment 2	Substrate A
B_T1	Treatment 1	Substrate B
B_T2	Treatment 2	Substrate B
7.2	DIGE Tutorial
The sample gels used in this tutorial come from an experiment that aims to study protein expression changes between control and treated groups of bacterial cultures.
In this study, eight sample lysates were prepared; four were derived from treated bacterial cultures and four were derived from un-treated (control) bacterial cultures. Aliquots from each sample were taken and pooled to prepare a standard sample (pooled internal standard). The samples were run on four gels. The control and treated samples were alternatively labeled with Cy3 or Cy5 (dye swap) to avoid dye-bias. The standard was labeled with Cy2 and run on each gel in the experiment. A preparative gel, including reference markers, was run on the pooled sample and post-stained with a Fluor total stain to pick spots and identify them by mass spectrometry (MS).
Gel	Cy2	Cy3	Cy5
Gel 01	Pooled internal standard	Control A	Treated A
Gel 02	Pooled internal standard	Treated B	Control B
Gel 03	Pooled internal standard	Control C	Treated C
Gel 04	Pooled internal standard	Treated D	Control D
7.3	Coverage Tutorial
The sample images used in this tutorial come from a 2D Differential In Blot Electrophoresis (2D-DIBE) experiment aimed at assessing coverage, i.e. the percentage of immunodetection that an antibody reagent offers for a population of HCPs. 
An E. Coli HCP sample was pre-labeled with Cy5TM and separated on a 2D gel. Protein was transferred from the gel to a membrane. The blot was then incubated with an anti-E. Coli HCP polyclonal antibody and a Cy3TM pre-labeled secondary antibody, and imaged for Cy5 and Cy3 to detect HCP antigen and anti-HCP antibody signals, respectively. The experiment was carried out in duplicate (Blot1 and Blot2).
Blot	Cy3	Cy5
Blot1	anti HCP antibody	HCP antigen
Blot2	anti HCP antibody	HCP antigen
8	Uninstall Instructions
To uninstall the Melanie package:
•	In the Windows search box, type “program” and choose Add or remove programs.
•	Select Melanie in the list.
•	Click Uninstall.