Analyse Western Blot and 2D gel images with Progenesis SameSpots

Did you know Progenesis SameSpots can be used to characterise proteins as well as measure differential expression analysis on your 2D gels? Many customers use Progenesis SameSpots to quantify protein expression changes, but identifying and validating the discoveries you make is often a requirement of further research funding or publishing data.

We’ve written an application note with Dr Meng Zhang (Northumbria University) to demonstrate how you can quantify expression and characterise proteins within the same experiment using Progenesis SameSpots.

In this case, we compared a Coomassie stained 2D gel with a Western Blot. But you can also apply the same approach to detect the presence of phosphoproteins or glycoproteins on your 2D gels.

Application Note – Compare Western Blots and 2D gels to characterise proteins
Download (0.99MB)

The Experiment & Analysis Set-up

The aim of our experiment was to detect the presence of a hex-His tagged protein over expressed in E. Coli P2 (ZP_02929368). Two duplicate 2D gels were run; the first was stained with Coomassie blue to show effective separation was achieved. The second 2D gel was blotted using Mouse Monoclonal Anti-polyHistidine primary antibody, secondary anti-body applied and visualised. The blotted 2D gel was then Coomassie stained to check how effective protein transfer had been and an image generated.

In this case, direct comparison of the very different spot patterns from all three images was achieved using two key features:

multiplex group1. “Multiple stains per gel without internal standards” experiment set-up

2. Creating a multiplex structure (shown left) to facilitate alignment

 

Results

The 2D gel image generated from Coomassie blue staining to show  effective separation was achieved was chosen as the Reference Image. The blotted 2D gel image (stained post-blotting) and the Western Blot image were aligned to this reference and the  pattern of detected spots applied to them all.  We correctly detected the presence and location of the His-tagged protein by comparing spot patterns across all images in this way.

hex-his-2d-spot-vs-western-blot

    2D gel stained post blotting  Western Blot Separate 2D gel stained for protein

Comparing spot patterns generated by aligning and detecting two duplicate 2D gels with a Western Blot. For full details see the application note. Squares highlight the His-tagged protein of interest on all three images generated during the experiment. The solid green line connects the spot of interest in the blot with the same spot on the 2D gel (stained post-blot) that it was blotted from. The dotted green line connects the spot of interest on the blot with the same spot on the duplicate 2D gel, stained for protein only and not blotted.

Try it for yourself

If you have a secondary staining application you’d like to try, download Progenesis SameSpots and use your 6 free, trial image licences to test it out. Or contact us and we can look at your data with you in more depth online. This will show the best way to set up your images for comparing the different spot patterns you may have.

I’ll be following this post up with a look at other applications, which go beyond discovery research, using Progenesis SameSpots to analyse secondary stains of the same 2D gel. In the meantime, if you have any questions about how to set up image analysis of 2D gels vs. Western Blots or other types of stains, you can post a comment here or ask us a question.

One Comment

  1. GATHONI KAMUYU
    Posted May 29, 2015 at 3:24 pm | Permalink

    Hello,

    I am conducting 2DE gels for western blotting and probing with patient immunoglobulins. I am interested in identifying differentially recognized spots by mass-spectrometry. The actual experiments involve separation of lysate proteins from P. falciparum in 3 2DE gels. One will be silvertstained, the remaining 2 transferred to a blot, 1 probed with patient IgG and 2nd probed with Negative control. I am using a FITC labelled secondary antibody so the images are flourescent as opposed to an exposed Xray film. Any tips on data analysis. I am also using a MAC and was looking to download your free trials, do you have MAC versions.

    Kind Regards
    Gathoni

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