SpotMap

Technical Specifications

SpotMap gives you the tools you need to quickly and reliably analyse HCP coverage using 2D gel and Western blots.

 

PC Specifications

Windows XP, Windows Vista, Windows 7, 8 and 10. Performance depends on processing power.

SpotMap supports multi-core CPUs and 64bit versions of Windows, which are recommended for maximum performance

SpotMap analysis features

What is Image QC?

When adding images to the experiment they are automatically checked for quality and any issues/notes are highlighted for you immediately before any further analysis is attempted.

 

What problem does it solve?

Poor image quality can make analysis of your 2D gels and Western blots difficult and reduce the objectivity of your results.

 

What are the benefits of using high quality images?

Quantification of spot parameters may seem less important when only looking at spot numbers and coverage results however good quality images also improve:

  • Spot Detection
  • Filtering options when selecting small/low spots to remove
  • Definition of spots on the blot (no saturation)
  • Automatic Alignment algorithm

Contact us for more information on image quality and capture.

Image QC

Quality issues with the images are clearly identified to give you feedback on your images before you start your analysis.

What is the Image Editor?

It is a tool you can access from the software if your images require minor changes (flipping etc.) then you can edit them without changing the original images

 

What problem does it solve?

Images which are different in size, shape and orientation are hard to compare directly. Simple edits to fix this can be completed within the software. Editing in a different software program may cause data to be lost from the image, reducing the objectivity of the results. Using the editing tools available in SpotMap minimises data loss.

 

What edit options are available?

 

  • Flip images horizontally and vertically
  • Rotate images 90 degrees clockwise and anti-clockwise
  • Invert image
  • Translate image
  • Crop image
  • Rotate image a variable angle
  • Resize images to same size
image edit tools

 

 

Basic image edits can be completed in the image upload page, including: invert image, flip horizontally, flip vertically and crop. Further editing is available in the image editor tool.

Image editor

Further image edits can be completed in the image editor.

What is Alignment?

Alignment takes two images and overlays them in the same coordinate space so that a spot on the 2D gel will be in the same location as the matching spot on the Western blot.

 

What problem does it solve?

Alignment makes it possible to accurately compare images by removing any positional variation introduced during the gel running, blotting and imaging process.

 

Why is alignment better than spot matching techniques?

Using separate spot detection on the blot and then matching back to spots on the gel is very inefficient. Spot detection on the blot is harder and it can be difficult to compare back to the gel. Alignment makes it possible to overlay spots from the gel onto the blot.

 

How is alignment different to warping?

Warping and alignment both aim to remove the positional variation introduced during the blotting process. However warping is only accurate to the spot level whereas alignment is accurate to near pixel level.

 

Do I need to align DiGE images?

Alignment is not necessary for DiGE images. However, for low MW proteins there may be a shift due to the different dye, in this case alignment can be used.

 

Can I “fix” alignment after spot detection?

Yes this is possible. If you see a problem whilst editing spots then just come back to this stage and add/remove vectors as required.

 

How does Alignment work?

Automatically generated or manually added vectors between features on each image are used to create a map between the images. If the blot has clear spot patterns then this can be fully automatic using our fast alignment algorithm.

image unaligned

 

 

 

 

 

image aligned

 

 

The alignment can be visibly checked as the Western blot image (green) should be perfectly overlaid to the 2D gel (magenta). The top image shows the gel and blot unaligned, the bottom image shows the images aligned.

What is Spot Coverage?

Western blot analysis in SpotMap is used to look at the coverage of the Western blot antibody. This is the percentage of gel spots that appear in the blot.

 

How do we calculate the spot coverage?

Coverage equals the number of spots on the blot/number of spots on the gel. Sometimes spots only appear on the blot and have not been detected on the gel. These are counted separately as they may be of interest. In our calculation these are also counted as spots on the gel as proteins must exist on the original gel to create an antibody reaction.

 

What does Automatic Spot Detection do?

It takes the gel image and using advanced imaging techniques it creates a series of spot shapes which are used to create spot measures.

 

Why are spots copied from the gel to the blot and not separately detected on the blot?

Western blots are often fuzzier than stained gels with spots that have bloomed due to way antibodies are detected on the blot. Algorithms designed to detect spots on gels often fail on blots due to this difference. We therefore detect on the cleaner gel and copy the spot shapes over to the blot after alignment. This really helps the user see where the spots exist on the blot.

 

Why edit spots?

Sometimes the automatic spot detector results need to be improved. Spot shapes around the edge can be removed. Spot shapes in the middle can be added/deleted/merged or split. Our tests have shown that you can have different levels of editing (little or a lot) and still get similar coverage results. It is key to be consistent so that you get similar results across experiments.

 

Why select spots using clauses?

The filter option is usually used to select spots you may wish to “remove” or make “unique” in the gel or blot. Use a clause based on an individual spot will select spots similar to this spot.

Coverage and spots

 

 

 

 

Coverage = Number of spots present on Western blot / Total number of spots present x 100.

Blot Image

Western blot image with the complete spot map of all protein spots present on the gel image.

 

 

What does the review screen show?

All the results from the project can be displayed or exported from the review page.

 

What can you export using Data Access?

 

  • Spot Detection parameters
  • Image QC results
  • Gel, blot and combined images with all possible overlays
  • Coverage results
  • Spot numbers

What can you show on the image?

  • Spots common to the gel and blot
  • Spots unique to the gel
  • Spots unique to the blot
  • User selected interesting spots
  • User added annotations
  • Combined image of gel and blot in false colours
Complete experiment The summary screen allows you to review your data,  highlight spots of interest and annotate the spot map.

 

 

 

data access

 

Drag and drop data direct from the Data Access tool into your reports and presentations.


FAQs

Can SpotMap Calculate Spot Coverage?

Yes. A spot map is created of the fully resolved host cell protein, determining the presence of spots expressed on the blot is then used to calculate the relative coverage of Western blot vs the 2D gel.

Can SpotMap Check Image Quality?

Yes. When adding images to the experiment they are automatically checked for quality and any issues/notes are highlighted for you.

Can SpotMap Edit My Images?

Yes. If your images require minor changes (flipping etc.) then you can edit them within the software without changing the original images.

Can SpotMap Account For 2D Gel or Blot Distortions?

Yes. Alignment makes it possible to accurately compare images by removing the positional variation introduced during the electrophoresis and blotting process.

Can SpotMap Export My Results?

Yes. All the calculations, spot numbers, images and data can be easily exported from the program.

Coverage

Get technical support direct from the people who developed your software. We will work personally with you to ensure you are fully trained to get the most out of SpotMap.

Got any questions? Get in touch with us today!

See SpotMap in action

SpotMap analysis report

See the workflow of SpotMap

Workflow