Delta2D Report: Blotting
for Project 'Coverage Report'
Project Properties
| Author | DECODON Team | Project Creation Date | Tue Nov 01 09:20:00 CET 2016 | ||
|---|---|---|---|---|---|
| Use Internal Standard | no | Pool | C:\data\Blot report | ||
| Description | Comparison Totalprotein and 2D western blot | ||||
| Report created by user "bielz" with Delta2D, Fri Nov 04 10:16:26 CET 2016. Please let us know if you have any comment or suggestion using our contact form. | |||||
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Sample to Gel Assignment
| Group Name | Gel | Channel | Removed Speckles | Gel Image Name |
|---|---|---|---|---|
| Total Protein | Not assigned | Silver | black: 8; white: 0 | Bet v 1 Silverstain |
| 2D WB | Not assigned | Not assigned | black: 8; white: 6 | WesternBlot-Bet v 1 |
| Author: DECODON Team |
Experimental Setup
Used approach: This is a 'Gel - Membrane / Western Blot' project.
The coverage between each Gel image and Western Blot image is computed with this formula:
| Coverage (COV2) = | No. spots Western Blot Image | * 100% |
| No. spots Total Protein Image + No. unmatched spots Western Blot Image |
The coverage among the same kind of image (Gel, Western Blot) is computed with this formula:
| Coverage (COV1) = | No. matched spots | * 100% |
| No. matched spots + No. unmatched spots Image 1 + No. unmatched spots Image 2 |
Direct Warped Pairs (Warp Relations)
These image pairs have been warped to eliminate gel distortions.
| Warped Image | Warped to Image | Warp Mode | Match Vectors |
|---|---|---|---|
| Fused Image using Max Intensity at 01_11_2016 11_35_38_845 | Bet v 1 Silverstain | global | 1 |
| WesternBlot-Bet v 1 | Bet v 1 Silverstain | exact | 10 |
| Author: DECODON Team |
Fused Images from Group 'Fused Images'
Fused Image Name: Fused Image using Max Intensity at 01_11_2016 11_35_38_845
- Master Gel Image: Bet v 1 Silverstain
- Input images: Bet v 1 Silverstain, WesternBlot-Bet v 1
- Input images have been processed before fusion by applying: Remove Background, Amplitude Rescale
- Fusion type: Max Intensity
- Image sizes have been adjusted to: Common Region
- Fused image has been processed by applying: Amplitude Rescale, Remove Background
| Author: DECODON Team |
Transferred Spot Pattern
| Transfer Origin (Fused Image): | Fused Image using Max Intensity at 01_11_2016 11_35_38_845 |
|---|---|
| Spot Detection Parameters | |
| Local Background: | 125 |
| Average Spotsize: | 41 |
| Sensitivity: | 20.0 |
| # of originally detected spots: | 916 |
| # of manually added spots: | 1 |
| # of spots after spot editing: | 917 |
| # of canceled spots: | 755 |
| # of transferred spots: | 162 |
| Author: DECODON Team |
Fused Image 'Fused Image using Max Intensity at 01_11_2016 11_35_38_845'
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Detected spots.
Canceled spots.
Spot edit markers.
| Author: DECODON Team |
Image Pairs
Image pairs have been created in accordance with the 'Gel - Membrane / Western Blot' structure of this project.
Unmatched spots among filter settings in Delta2D.
Dual View for 'Bet v 1 Silverstain' and 'WesternBlot-Bet v 1'
| matched spots: | 61 of 162 (blue) |
Coverage: 41.98% |
|---|---|---|
| unmatched spots on Bet v 1 Silverstain: | 94 of 162 (green) | |
| unmatched spots on WesternBlot-Bet v 1: | 7 of 162 (red) |
| Author: DECODON Team |
Coverage Table
Group columns are ordered in the same way as the group rows, i.e. the name of the image in column 2 of a certain group can be taken from the second row of the said group.
| Group | Total Protein | same as row group | |
|---|---|---|---|
| Image | 1 | 1 | |
| Total Protein | 1 'Bet v 1 Silverstain' | - | - |
| 2D WB | 1 'WesternBlot-Bet v 1' | 68/162 41.98% | - |
| Author: DECODON Team |
Results
Reproducibility for Image Pairs in Groups
| Group | N | Mean | RSD |
|---|---|---|---|
| Total Protein | 0 | VV N.A. % | VV N.A. % |
| 2D WB | 0 | VV N.A. % | VV N.A. % |
Statistics on Relative Coverage for Group Pairs
| Total Protein Group | Western Blot Group | N | Mean | RSD |
|---|---|---|---|---|
| Total Protein | 2D WB | 1 | 41.98 % | 0.00 % |
Remarks
Please note, that this Blotting report is just an example and as such was created from different Gel images than the rest of the project.
| Author: DECODON Team |
About the Report
Blotting Report Attachment (page 1 of 2)
Analysis Procedure
- The project may contain up to 9 groups (plus fused image group and internal standard group), further groups will be ignored.
- Each group may contain up to 6 images, further images will be ignored.
- Image names are not longer than 35 characters, gel numbers not longer than 2 characters.
- The group containing fused images is named 'Fused Images'.
- A group called 'Internal Standard' will be ignored as this group is assumed to contain just internal standard images.
- Spot transfer to all images must be done from the same fused image.
Experiment Structure
Westernblots with multi fluorescence, maximal 3 Westernblot dyes. Total-Protein group must be the first group in the project.Image pairs:
- each Total Protein Image with one Westernblot Dye N (N: 1...3)
images in all groups must be sorted the same way to enable an appropriate assignment of each western blot image to the respective total protein image - all possible pairs within each group
- Special coverage formula for image pairs Total Protein Image - Westernblot Dye
Example:
COV1 = Coverage formula for Gel - Gel approach
COV2 = Coverage formula for Total Protein - Western Blot approach:Coverage (COV1) = No. matched spots * 100% No. matched spots + No. unmatched spots Image 1 + No. unmatched spots Image 2 - A number of 1 (k) group 'Total Protein' and 3 (l) groups with 'Westernblot Dyes' with 6 (m) images each results in 6*3+4*(6*6-6)/2=78 image pairs, i.e. the same number of report pages, general: m*l+(k+l)*(m*m-m)/2.
| Coverage (COV2) = | No. spots Western Blot Image | * 100% |
| No. spots Total Protein Image + No. unmatched spots Western Blot Image |
Blotting Report Attachment (page 2 of 2)
Report Content
- Section 'Sample to Gel Assignment':
- The section shows a table summarizing the sample to gel assignment used in this Blotting Report.
- Section 'Experimental Setup':
- The coverage formulas are automatically applied.
- Section 'Fused Images from Group 'Fused Images'':
- On one page a maximum of 6 fused images is described (configurable).
- Section 'Transferred Spot Pattern':
- The number of spots after spot editing is the sum of originally detected spots and manually added spots.
- The number of transferred spots is the difference between spots after spot editing and canceled spots.
- Section 'Fused Image 'Fused Image using Max Intensity at 01_11_2016 11_35_38_845'':
- The image shows all spots and spot edit markers (if available).
- Canceled spots are shown with dotted spot boundaries.
- Section 'Image Pairs':
- Includes data for the interesting image pairs for which the 'Coverage' table includes values.
- Images show spot boundaries for interesting spots only, not for canceled spots.
- Matched Spots are shown in blue, unmatched in green or red, respectively.
- Section 'Coverage Table':
- Coverage is calculated based on the formula as shown in the examples above.
- Coverage table is split after a maximum of 2 leading columns and 14 value columns and/or 2 leading rows and 40 value rows (configurable).
- Groups will not be split.
- To reduce the width of coverage tables a legend can be introduced.
- Section 'Results':
- 'Reproducibility for Image Pairs in Groups' (1) and 'Statistics on Relative Coverage for Group Pairs' (2) are always based on the respective existing coverage values.
- 'Mean' is the mean of coverage value of each image pair
- 'RSD' (relative standard deviation) is the 'corrected sample standard deviation' (as defined in https://en.wikipedia.org/wiki/Standard_deviation), expressed in %.
- 'N' is the number of image pairs (1) or the number of replicates (2)
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