SameSpots: 2D Gel and 2D-DIGE Analysis Software for Differential Protein Expression

Why SameSpots is the Most Reliable 2D Gel Analysis Software for Proteomics

SameSpots (previously Progenesis SameSpots) overcomes the common problems you face when using 2D gel electrophoresis proteomics work to measure differential protein expression. These include a lack of reproducibility in your results, hours of tedious spot editing and alignment and a lack of confidence in the resulting statistical significance of protein expression changes. SameSpots offers all the functionality you need to analyze experiments consisting of conventional 2D gels, 2D Western Blots, 2D-DIGE and 2D-DIBE gels all within a single package so that you can fully exploit the advantages offered by multiplexing with no upsells or add-ons. Our software offers:

• Simplicity. You are guided through each step of the workflow, new users can get up and running with minimal training.
• Speed. Typical analysis times of less than 5 minutes per gel with single-stain or 2-DE DIGE images.
• Objectivity. A consistent analysis approach that is proven to give reproducible results between different users.
• Statistical power. Our next generation pixel-level image alignment algorithms deliver 100% matching with no missing values in your data for valid multivariate statistics.
• Expression clustering. Group proteins by expression profile with built-in hierarchical clustering and dendrograms.
• 21 CFR or GMP-compliance. When combined with our AuditSafe software, allows users to meet and even exceed FDA 21 CFR part 11, EudraLex Annex 11, GAMP4 or more general GMP standards/ALCOA principles for compliance within regulated or QC environments, from captured image all the way through to final analysis and report production.

SameSpots is tested and compatible with Windows 10 and 11.

Our SameSpots software is trusted by the world’s leading educational, industrial and pharmaceutical companies.

Bio-Rad named TotalLab its preferred alternative to PDQuest

When Bio-Rad discontinued PDQuest, they did not leave their customers to find a replacement on their own. They evaluated the options and wrote to their users naming TotalLab.

“The software packages we are recommending are SameSpots and SpotMap from TotalLab Limited, our preferred alternative vendor for 2D analysis software.”

Mona Chin, Global Product Manager, Bio-Rad Laboratories Customer announcement on the discontinuation of PDQuest, 2018 Read Bio-Rad’s announcement in full That recommendation covered SameSpots for 2D gel and 2D-DIGE analysis and SpotMap 2D for HCP coverage. It still stands.

What SameSpots does

Two-dimensional gel electrophoresis separates proteins by isoelectric point and then by molecular weight, producing a 2D protein gel image of hundreds or thousands of spots. Comparing those spots across gels is where most of the time, and most of the error, in a 2D proteomics experiment lives. SameSpots exists to remove both. If you are new to the technique, start with our guide to 2D electrophoresis: Guide to 2D electrophoresis

Every spot matched on every gel, with no missing values

Most 2D gel analysis software detects spots on each gel separately and then tries to match them. Spots that are detected on one gel and missed on another become missing values, and missing values break your statistics. SameSpots works the other way around. It aligns every image to a reference first, then detects one spot pattern across the whole experiment. The result is 100% spot matching: every spot has a measurement on every gel, and your statistical tests run on a complete dataset.

Analysis in under five minutes per gel

Automatic alignment and whole-experiment spot detection remove the manual spot editing that dominates other packages. Labs routinely report analysis that used to take days finishing in a few hours. A full experiment of 24 images can be analyzed in approximately one hour, and new users are typically productive after less than two hours of training.

Statistics built in, designed around your experiment

One-way ANOVA, two-way ANOVA, repeated measures ANOVA, principal component analysis, expression profiles, interaction plots, scatter plots and descriptive statistics are all built in. You choose the design that matches how you ran the experiment (see Experimental designs below) and SameSpots applies the right test. There is no export to a separate statistics package and no re-import of results. Our guide to p-values, FDR and q-values explains how to read the results.

One license covers 2D-PAGE, 2D-DIGE, 2D Western blots and 2D-DIBE

Conventional single-stain gels, fluorescent multiplexed DIGE gels, 2D Western blots and 2D-DIBE are all analyzed in the same software under the same license. DIGE is not a separate module and is not an extra purchase. SameSpots is vendor-neutral: it works with images from any scanner or imager and supports all common stains and industry-standard image formats.

2D-DIGE analysis software, without a separate DIGE module

2D-DIGE labels up to three samples with Cy2, Cy3 and Cy5 fluorescent dyes and runs them on a single gel, which removes gel-to-gel variation from the comparison. SameSpots fully supports 2D-DIGE analysis, including experiments run with a pooled internal standard, and works with saturation labeling workflows as well as minimal labeling. Because every spot is matched across every gel and every channel, DIGE experiments in SameSpots do not suffer from the missing data problems that make other packages hard to use for multi-gel DIGE designs. Mike Dunn, Professor of Biomedical Proteomics at the UCD Conway Institute, moved his lab to SameSpots for exactly this reason (his words are in the testimonials below). To understand when DIGE is worth the extra cost over conventional 2D-PAGE, read our comparison: 2D-DIGE vs 2D gel electrophoresis Deciding between gels and mass spectrometry for your project? Read 2D-DIGE vs LC-MS/MS: when to use each.

How the SameSpots workflow runs

SameSpots walks you through the analysis in a fixed order. Each step has to be completed before the next one opens, which is how it keeps different users in the same lab producing the same answer from the same images. Our step-by-step guide, how to analyze 2D gels with SameSpots, covers each step in more detail.

1. Image quality control

When you add images to an experiment, SameSpots checks them automatically before any analysis starts. It flags bit depth (16-bit images are strongly recommended for quantitative work), saturation (overexposed spots cannot be quantified accurately), inconsistent resolution or file size across the dataset, and inverted or misoriented images. Cropping, rotating, flipping, scaling and inverting tools are built in so you can fix problems without leaving the software.

2. Automatic alignment

Alignment removes the positional variation that is inherent to 2D electrophoresis. SameSpots uses a pixel-level image alignment algorithm that warps each gel image so its spots superimpose precisely on the reference gel. Alignment is automatic, with manual vector placement available for difficult regions, and the alignment can be reviewed and adjusted at any time. This step is what makes 100% matching possible. The result is that every spot you compare is the same spot on every gel, so a difference between conditions is a real change in abundance, not a matching error.

3. Spot detection across the whole experiment

Once the images are aligned, SameSpots detects a single spot pattern and applies it to every image. Every spot outline is therefore identical across the experiment, and an edit made to a spot on one gel propagates to that spot on every gel at once. There is no per-gel spot editing and no spot matching step. What you get is one list of protein spots for the whole experiment, each with a volume on every gel, ready to test for differential expression.

4. Review and normalization

Variation in loading, staining and imaging between gels is corrected by normalization. SameSpots expresses every spot as a ratio to the same spot on a reference (an automatically chosen reference gel, or the Cy2 internal standard on the same gel in DIGE) and scales each gel by the robust mean of its log ratios, with outliers excluded. A graph for every gel shows the ratios, the factor and the estimation limits, so a problem gel is obvious at a glance. You can then filter and tag spots, review the 3D view of any spot on any gel, and exclude artifacts before statistics. Once normalized, the fold change for every spot compares like with like across gels, stains and scanning sessions.

5. Statistical analysis

Choose your experimental design and SameSpots runs the appropriate ANOVA, then lets you explore the results with principal component analysis, expression profiles, interaction plots and scatter plots. Spots that change significantly can be flagged with the quick-tagging system so you can prioritize candidates for identification. SameSpots reports p-values, q-values and power for every spot; our guide to p-values, FDR and q-values explains what each one tells you. The output is the list a paper is written from: which protein spots are up-regulated, which are down-regulated, by how much, and how confident you can be in each one.

6. Spot picking and protein identification

Generate picking lists for spot-picking robots, including the Cytiva Ettan Spot Picker, or manual picking templates for excision by hand. Protein identifications from mass spectrometry can be imported back into SameSpots from MASCOT and from custom databases, so your final report links each spot to its identity and its statistics. pI and MW calibration is supported by defining known markers within the gel images.

Experimental designs SameSpots supports

Good 2D gel statistics start with the experimental design: which samples are replicates, which are independent, and which factors you are testing. SameSpots supports the three designs that cover the large majority of proteomics experiments, and applies the matching test automatically.

Between-subject design (one-way ANOVA)

Independent samples in two or more groups, for example treated versus control, or three disease stages. Each gel is a biological replicate from a different subject. This is the most common design in clinical, agricultural and microbial proteomics.

Within-subject design (repeated measures ANOVA)

The same subjects measured under more than one condition, for example the same patients before and after treatment, or the same cell line at several time points. Because each subject acts as its own control, this design removes subject-to-subject variation and is far more sensitive than a between-subject design of the same size. Repeated measures ANOVA is built into SameSpots; many 2D gel packages do not offer it.

Two-way ANOVA design

Two factors tested at once, for example genotype and treatment, or substrate and temperature. Two-way ANOVA tells you the effect of each factor and whether the two factors interact. Interaction plots in SameSpots make those interactions visible spot by spot.

Built for regulated and non-regulated labs alike

Academic labs use SameSpots as it comes. Labs working under GMP, or submitting data to the FDA or EMA, add AuditSafe, TotalLab’s compliance module. AuditSafe adds audit trails, electronic signatures, granular user permissions and image authenticity verification to SameSpots so that the whole analysis, from captured image to final report, meets FDA 21 CFR Part 11 and EU Annex 11 requirements. Many alternative 2D gel analysis tools have no equivalent compliance pathway. Learn more about AuditSafe If your 2D gel work is host cell protein antibody coverage for a biologic, rather than differential expression, you want SpotMap 2D. It was built for that specific assay and is the second product named in Bio-Rad’s announcement. SpotMap 2D for HCP coverage analysis

Who uses SameSpots

SameSpots has been in continuous development since 2008, when it was launched as Progenesis SameSpots. Today it is used by university proteomics groups, core facilities, pharmaceutical and biotechnology companies, agricultural and food research institutes, and teaching labs running 2D gel practicals for undergraduate and master’s students. It has been cited in more than 200 peer-reviewed publications, and an independent published comparison found that SameSpots outperformed two competing packages in matching accuracy. Independent comparison: 2D DIGE image analysis by DeCyder versus SameSpots

Published research using SameSpots

SameSpots has been cited in more than 200 peer-reviewed publications across proteomics, clinical research, agriculture, microbiology and food science. Browse the full list of SameSpots publications.

Powered by Bioz See more details on Bioz

Case Studies

Four labs, four sets of results. Read all of them on one page: SameSpots case studies.

Case Study: University of Texas Medical Branch, Biomolecular Resource Facility

The University of Texas Medical Branch, Biomolecular Resource Facility is one of only seven sites within the USA that receive multi-million dollar state funding by the US Department of Health & Human Services. “The fundamental concept (image alignment) of SameSpots permits us greatly increased confidence in quantification, and this is enhanced when performed on gels obtained by our saturation fluorescence strategy. The statistical package further enhances confidence in our quantitative studies. In addition SameSpots has increased our throughput – with the demand we experience, this results in tremendous savings in manpower and expense over a year. With the proper tools and attention to detail proteomics can reap enormous benefits whatever your field of biological study. SameSpots is one of the tools that are indisputably required — providing the image and subsequent statistical analyses that are critical to a conclusive and compelling proteomics study.” John E. Wiktorowicz, Ph.D. Associate Professor, Dept. Biochemistry & Molecular Biology Director, Proteomics Section

Case Study: Instituto di Ricerche Farmacologiche “Mario Negri”

Founded in 1963, The Instituto di Ricerche Farmacologiche “Mario Negri” is a non-profit independent scientific organisation for biomedical research and education. “In the Environmental Health Sciences Department we investigate the effect of environmental factors on human health, focussing on the survey of environmental contaminants, the assessment of human exposure with related health. “We have been using SameSpots in our research since January 2007. The software has drastically simplified our proteomics workflow and opens up new avenues for the exploration of the data obtained” “Since using SameSpots the speed of our analysis has been drastically improved. We align our gels accurately within 5 mins per gel and then run the SameSpots analysis. We don’t usually perform any correction of spot detection which results in fast, robust and very reproducible analysis. This has an immediate positive effect on the quality and confidence in our results.’’ Dr Roberta Pastorelli, Head, Protein and Gene Biomarkers Unit Laboratory of Molecular Toxicology

Case Study: Cardiovascular Sciences Research Centre, St. George’s University of London

The Cardiovascular Sciences Research Centre, St. George’s University of London, is a multidisciplinary group which amalgamates clinical, surgical and basic science research expertise. The mission of the Research Centre is to understand mechanisms of cardiovascular disease. “The main difference is the speed at which we can analyze our gels. Using SameSpots, we are able to analyze our gels in a much shorter time frame, with very little manual intervention required. Initial analysis that could take days before can now be achieved within a few hours, obviously depending on the number of gels. The results we are obtaining are extremely robust and reproducible. The matching is excellent, and hardly any manual spot editing is required. Also, the inbuilt statistics is user friendly, giving both q values, which are extremely useful to have in addition to the p values, along with the power value. The workflow is straightforward and logical, and visually appealing.” Dr. Ayesha De Souza, Senior Research Fellow, Cardiac Proteomics, Cardiac and Vascular Sciences, St. George’s University of London, London, UK.

Case Study: Centre Paul Papin

The Centre Paul Papin is a hospital specialized in cancer treatment and cancer research. The Centre Paul Papin collaborates with the Plateforme technologique d’Angers which offers, among other things, services in proteomics to public laboratories and industry. “With our previous software, we experienced three major problems. Firstly, despite the time we spent disposing of many landmarks on the master gel, the software made many mistakes in alignment. Secondly, when a spot was missing on the gel master, it was not detected and analysed. Finally, the software was not friendly to use and the analysis was laborious.” “SameSpots is so convivial and intuitive that it is a real pleasure to work with. The image alignment step makes us feel secure that we have one hundred percent matching and the additional statistical tools are perfectly designed for biologists who are not specialized in statistics. The development and improvement of SameSpots has been pleasing.” Prof Catherine Guette, Proteomics platform leader, Laboratoire d’oncopharmacologie, Nice, France

How SameSpots compares to other 2D gel analysis software

There are now very few actively maintained commercial packages for 2D gel differential expression analysis. Several that were widely used have been discontinued. This section is for labs deciding whether to switch, and what to switch to.

SameSpots vs Melanie

Melanie, from the SIB Swiss Institute of Bioinformatics and distributed by Cytiva, is the other actively developed commercial package. The two take different approaches. Melanie sells expression analysis (Classic and DIGE) and coverage analysis as separate modules; SameSpots covers 2D-PAGE, 2D-DIGE, 2D Western blot and 2D-DIBE in one license. Melanie offers one-factor and two-factor designs; SameSpots adds repeated measures ANOVA for within-subject designs. Melanie states that full 21 CFR Part 11 compliance requires additional controls beyond the software; SameSpots with AuditSafe provides the compliance layer itself.

Moving from PDQuest (Bio-Rad)

PDQuest is discontinued and Bio-Rad named TotalLab its preferred alternative vendor. Our migration guide covers how PDQuest concepts map onto SameSpots, what to expect from the first analysis, and how to bring across existing image sets. Why upgrade from PDQuest to SameSpots

Moving from DeCyder (GE Healthcare / Cytiva)

DeCyder is no longer developed. An independent peer-reviewed comparison found SameSpots more intuitive to use, with markedly less time needed for quantitative analysis, because alignment before detection replaces DeCyder’s time-consuming spot matching step. Why upgrade from DeCyder to SameSpots

Moving from Delta2D (DECODON)

If you are running Delta2D, the questions to ask are the same as for any package: how are missing values handled, which statistical designs are built in, is there a compliance pathway, and is the software still being developed and supported. SameSpots answers all four on this page. Ask us for a side-by-side on your own images.

Licensing, pricing and support

SameSpots licenses cover an entire computer with no restriction on the number of users, which suits shared instruments and core facilities. Single-computer licenses, discounted multi-computer bundles and enterprise or site-wide agreements are available, and prices can be quoted in GBP, USD and EUR. SameSpots runs on Windows 10 and Windows 11. It is not available for macOS. Buy direct from TotalLab for the latest version and the most direct support, or through our distributor network if you need to purchase locally or in your own language. Find your local distributor Request your free trial

How to cite SameSpots: SameSpots (TotalLab Limited, Newcastle upon Tyne, UK), with the version number you used. Citing the software in your methods helps other labs find it.

Frequently asked questions

What is SameSpots used for?

SameSpots is 2D gel analysis software for proteomics. It aligns, detects, matches and quantifies protein spots across 2D-PAGE, 2D-DIGE, 2D Western blot and 2D-DIBE images, then runs the statistics needed to identify proteins whose expression differs between conditions. The full workflow is described in how to analyze 2D gels with SameSpots.

Is SameSpots an alternative to Melanie?

Yes. SameSpots and Melanie are the two actively maintained commercial packages for 2D gel differential expression analysis. SameSpots covers conventional gels, DIGE and blots in a single license, includes repeated measures ANOVA, and offers a 21 CFR Part 11 compliance pathway through AuditSafe. A comparison table is on this page.

Can I use SameSpots to replace PDQuest?

Yes. When Bio-Rad discontinued PDQuest they named TotalLab their preferred alternative vendor and recommended SameSpots to their users. A migration guide is available on this page, and the announcement is covered in full in PDQuest discontinued: Bio-Rad recommends TotalLab.

Does SameSpots match all spots across all images?

Yes. SameSpots guarantees 100% spot matching with no missing values across all images in your experiment, because it aligns the images first and then detects one spot pattern across the whole experiment.

Can I analyze 2D-DIGE images?

Yes. SameSpots fully supports 2D-DIGE analysis for experiments using Cy2, Cy3 and Cy5 fluorescent dyes, including experiments run with a pooled internal standard. DIGE support is included in the standard license. If you are deciding whether DIGE is worth the extra cost, read 2D-DIGE vs 2D gel electrophoresis.

What statistics are built in?

One-way ANOVA, two-way ANOVA, repeated measures ANOVA, principal component analysis (PCA), expression profiles, interaction plots, scatter plots, descriptive statistics and power analysis. SameSpots reports a p-value, a q-value and the statistical power for every spot; our guide to p-values, FDR and q-values explains how to interpret them.

How long does analysis take?

A typical gel takes under five minutes. A full experiment of 24 images can be analyzed in approximately one hour.

How does alignment work?

SameSpots identifies corresponding spots between each image and the reference image, then warps the image so that its spots precisely superimpose with those in the reference coordinate space. This removes the positional variation inherent to 2D gel electrophoresis before any spot is detected. Read more in 2D gel image alignment explained.

What is the reference gel?

The reference gel is the image that all other images in the experiment are aligned to.

Can I check image quality before analysis?

Yes. When images are added to an experiment they are automatically checked for bit depth, saturation, resolution and file size consistency, and inversion or orientation problems. Issues are highlighted immediately, before any analysis is attempted. Our guide to imaging 2D gels explains the scanner settings that avoid these problems in the first place.

Can I edit my images?

Yes. Cropping, rotating, flipping, scaling and inverting tools are built in.

How is different gel loading corrected?

Through normalization. Every spot is expressed as a ratio to the same spot on a reference gel, or to the Cy2 internal standard in DIGE, and each gel is scaled by the robust mean of its log ratios with outliers excluded. The statistics then run on log-transformed normalized volumes. See normalization in 2D gel and 2D-DIGE analysis for the methods compared.

Can I calculate pI and molecular weight?

Yes. Define known pI and MW markers within your gel images and SameSpots calibrates the rest.

Can I generate picking lists?

Yes. SameSpots exports picking lists for spot-picking robots, including the Cytiva Ettan Spot Picker, as well as manual picking templates. See 2D gel spot picking and protein identification for the full process from spot to identity.

Can I import mass spectrometry identifications?

Yes. Protein identifications can be imported from MASCOT and from custom databases and attached to the matching spots.

Can I export my results?

Yes. Data can be exported at every stage of the analysis in CSV format or as HTML/PDF output in a formal report. All images can also be exported for formal presentation as well.

Which image formats and imagers does SameSpots support?

SameSpots is vendor-neutral and works with images from any scanner or imager, in all common stains and industry-standard image formats such as .TIFF, .JPG, .PNG, .BMP etc. For the resolution and bit depth to scan at, see imaging 2D gels.

What operating systems does SameSpots run on?

Windows 10 and Windows 11. SameSpots is not available for macOS.

Does SameSpots do HCP antibody coverage analysis?

HCP coverage analysis is a different assay with different outputs, so TotalLab provides SpotMap 2D for it. SpotMap 2D and SameSpots are both named in Bio-Rad’s PDQuest announcement.

Is SameSpots 21 CFR Part 11 compliant?

With TotalLab’s AuditSafe module, SameSpots provides audit trails, electronic signatures, granular user permissions and image authenticity verification to meet FDA 21 CFR Part 11 and EU Annex 11 requirements.

Is there a free trial of SameSpots?

Yes. Request a free trial and evaluate SameSpots on your own 2D gel or 2D-DIGE images. Trial licenses are usually issued within one business day.

Is there a free version of SameSpots?

No. SameSpots is commercial software. A fully functional free trial is available, and multi-computer pricing is available on request.

Has SameSpots been used in published research?

Yes. SameSpots has been cited in more than 200 peer-reviewed publications. A reference list is maintained on the TotalLab website.

How long does it take to learn?

New users are typically productive after less than two hours of training. The fixed step-by-step workflow means there are no decisions to make about what to do next.

Resources

Try SameSpots on your own gels

Request a free trial and we will send a license, usually within one business day. Analyze your own 2D gel or 2D-DIGE images, run the statistics, and see the difference 100% matching makes to your results.