Dear colleagues
We could successfully extract and purify the Phycoerythrin from two Nostoc strains with four major steps viz. preparation of crude extract (Step I), 65% ammonium sulfate precipitation (Step II), dialysis (Step III) and anion exchange chromatography (STEP IV) using DEAE-Cellulose-11 and Acetate buffer.
Now we have problem in SDS-PAGE process. We could not find the bands on the gel.
Please if anyone knows help us.
Thanks a lot
Hi Bahareh, Please can you give detailed information about your SDS-PAGE,condition used and the amount loaded into the wells so that colleagues could provide a comprehensive suggestions to solve your problem. Thanks
Thank you so much for your reply.
After dialysis we prepare a range of pH (3.1 to 5.1) and purified them with chromatography.
The purified Phycoerythrin with different pH has been loaded on SDS page. We did the staining with different method.
However there are no bands. I am not sure if the band could visualize with gel doc or naked eyes.
Or when is better to load the Phycoerythrin on SDS page?
The quality of the pigments is very high. Can I load that on the first step? Is it destructed during the chromatography? What’s the best method for finding the bands?
Thanks alot
SDS-PAGE (Phycoerythrin from two Nostoc strains)?
You have used the same protocol as described in:
Extraction and purification of Phycoerythrin from Anabaena variabilis (CCC421)
Hillol Chakdar and Sunil Pabbi
Phykos 42 (1): 25 – 31 (2012)
First question: did you obtain the molecules spectra (Phycoerythrin) in your DEAE cellulose Step. This is a first indication of the purification/
I advise you to to enhance the growth culture as a scale-up protocol to optimize the bacterial production and the Phycoerythrin purification.
May be I will discuss with you a new efficient protocol if you present information’s about the Nostoc growth conditions and the spectrum.
actually in each step we obtain the molecular spectra.
its OK and in each step the quality was higher.
could you find the bands on SDS page? in which pH?
If i may ask you what % of SDS-PAGE did you use and your running buffer too?Did you check the band under UV transilluminator?So i suggest that you check that and you can also check the article attached to see example of the purification process and condition used on SDS-PAGE. Most pH is 7(0.005M Phosphate buffer)
I hope this will help you.
10% SDS-Page . do you think that the concentration is OK for the bands with 14 and 16 KDa?
Hi Bahareh,
I think to have a better resolution of your desired band and with 16-18kDa, I will suggest you use 12%SDS-PAGE(i.e 12% resolving and 4-5% stacking. You should be able to see your band if the right running buffer is used and you stain with Coomassie blue staining first and if you didn't see anything you further stain with Silver staining(more sensitive if the amount of protein loaded is small. You can also increase the volume loaded perhaps the amount loaded in each well is small to be detected.
All the best!
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