Author Topic: Deep Purple Stain  (Read 10543 times)

Andy

  • Guest
Deep Purple Stain
« on: October 23, 2003, 05:00:00 PM »
Has anyone used this new fluorescent stain from Amersham?  We routinely use Sypro Ruby but would love to save money and get better resolution.  Thanks for any response.

josef buelles

  • Guest
Re: Deep Purple Stain (Andy)
« Reply #1 on: October 28, 2003, 05:00:00 PM »
Yes I'ved used it for some tests. A good overview will give the attached comparison between Sypro Ruby and Deep Purple. Please contact your local sales office for price information ( code no. RPN 6305 or RPN 6306).

s.dirkx

  • Guest
Re: Deep Purple Stain (josef buelles)
« Reply #2 on: July 29, 2004, 05:00:00 PM »
There are very little comments about Deep purple or Sypro Ruby (fluorescent staining) here.
I'll try it next week, but I was hoping for some opinions about this staining.

LRC

  • Guest
Re: Deep Purple Stain (s.dirkx)
« Reply #3 on: August 01, 2004, 05:00:00 PM »
I used both Sypro Ruby and Deep Purple. Deep Purple gives a cleaner
background but with a more complicated procedure. However, I did not see how cheap it is and the higher sensitivity as it was said.

LRC


hansv

  • Guest
Re: Deep Purple Stain (LRC)
« Reply #4 on: August 02, 2004, 05:00:00 PM »
In our hands, and those of others of whom I have seen data, Deep Purple does not match an optimal Sypro Staining in terms of sensitivity. My guess would be that there is a twofold difference in sensitivity. For Sypro, we use a slightly modified procedure, which gives best results in our hands on 1.5 mm 2D Iso- or EttanDalt size gels (fixation in EtOH/HAc, overnight staining, washing in H2O). For Deep Purple we used the recommended procedure. However, as was already said, the background is much cleaner. Nevertheless our image analysis software (Progenesis) confirms the visual impression and detects more spots with Sypro. And, for saving money, I think neither of them is a good choice, because Amersham seem to have carefully adjusted the price of Deep Purple to get a close match to Sypro.
Best

Hans


hansv

  • Guest
Re: Deep Purple Stain (hansv)
« Reply #5 on: August 02, 2004, 05:00:00 PM »
Just to complete my previous comments, a comparison of the two dyes, using HeLa cells as a sample. Standard isoDalt 2D gel, 18 cm pH 4-7. Gels were run under our standard conditions (see papers by Hoving et al.) and scanned on the Fuji FLA-3000. Sypro staining as described, Purple as recommended by Amersham (incl. scanning). Images have been treated in Melanie, and both had contrast adjusted to the highest pixel (in the respective gel). So grayscales are not identical to make the signal-to-noise ratios comparable. The highest pixel (16-bit) in the Sypro images runs up to 7'000, the Purple to 3'000.

s.dirkx

  • Guest
Re: Deep Purple Stain (hansv)
« Reply #6 on: August 18, 2004, 05:00:00 PM »
I've tried Deep Purple, but was dissapointed in the sensitivity (compared to colloidal coomassie and silverstaining).

I can't find figure out what the problem is. I use the Typhoon 9200 for scanning (green laser 532 nm) with the 560LP or 610BP30-filter. I choose high sensitivity and 200 microns.

Anyone have some suggestions?!


hansv

  • Guest
Re: Deep Purple Stain (s.dirkx)
« Reply #7 on: August 18, 2004, 05:00:00 PM »
Does the difference in sensitivity look anything like our results? Maybe you could provide a picture comparison, with some indication of the loading? From everything I have seen in our own lab and in other labs, the expected outcome is that (under optimal conditions) Sypro is more sensitive than Deep Purple. The only explanation I have for the fact that the Amersham brochure shows the opposite, is that they used a suboptimal Sypro protocol. Although we did not do a direct comparison of Coomassie and Purple, it is hard to imagine that Coomassie is better. If that is the case, your settings/procedure might need modifications. The laser/filter settings we used (with the Fuji FLA_3000) are basically the same as yours. In my opinion a comparison with silver is not very informative, since with silver you can get anything you want in the absence of an endpoint.
Best wishes
Hans

keith

  • Guest
Re: Deep Purple Stain (hansv)
« Reply #8 on: August 20, 2004, 05:00:00 PM »
Dear hansv,

I'm sorry, I'm a beginner. Could you explain the last sentense: "In my opinion a comparison with silver is not very informative, since with silver you can get anything you want in the absence of an endpoint."  

Thanks very much


Tom

  • Guest
Re: Deep Purple Stain (hansv)
« Reply #9 on: August 21, 2004, 05:00:00 PM »
Hans, did you try the new protocol, relaesed very recently. There was a problem with the old protocol which used ammonia. But we found with the new protocol that the stain was much brighter than sypro ruby and more sensitivie, basically as claimed

hansv

  • Guest
Re: Deep Purple Stain (Tom)
« Reply #10 on: August 22, 2004, 05:00:00 PM »
Hi Tom

Thanks
No I did not. We did this comparison, soon after Deep Purple came onto the market. Is the protocol available on the Amersham website? We would love to try it to get an alternative to Sypro.

Thanks again

Hans


hansv

  • Guest
Re: Deep Purple Stain (keith)
« Reply #11 on: August 22, 2004, 05:00:00 PM »
Hi Keith

Thanks for your reaction. What I meant was that unlike Coomassie and Sypro, where at some point the staining is 'complete' (it does not get any more intense), with silver you can basically make a band or spot as intense as you want. Within certain limits of course. What happened in the past was actually the opposite (e.g. in papers about Sypro). People were showing that their stain was better than or as good as silver, without so much as standardizing the development time or even describing it. Doing that I can easily 'prove' that Coomassie is much more sensitive than silver, simply by stopping the silver staining early enough. But it is not very meaningful. For the same reason, it is pretty tricky to do a quantitative comparison using silver-stained gels, unless you can control and reproduce development times (and temperature of the reagents!!) within very narrow limits.
Hope this answers your question
Best
Hans


keith

  • Guest
Re: Deep Purple Stain (hansv)
« Reply #12 on: August 22, 2004, 05:00:00 PM »
Thanks Hans          

Keith


Tom

  • Guest
Re: Deep Purple Stain (hansv)
« Reply #13 on: August 22, 2004, 05:00:00 PM »
Hans

I can definitely recommend the new protocol for the Deep Purple. It comes with the product. I'm not sure if it is on their web page. We are getting sensational results with the new protocol. With the old protocol we found it was a bit mixed. Speaking to Amersham rep it appears there was an issue of controlling the pH that the new protocol has rectified this. Anyway, it is working beautifully for us and as simple as sypro.


Sjouke Hoving

  • Full Member
  • ***
  • Posts: 176
Re: Deep Purple Stain (Tom)
« Reply #14 on: August 23, 2004, 05:00:00 PM »
Can someone (from Amersham) post the pdf file with the modified protocol for the Deep Purple stain? Thanks in advance,

Sjouke

PS. I found it in the meantime on the site.

Modified by sjouke at 11:39 AM 8/24/2004