Hi Vincent, thank you for the answer! Do you know any "homemade" protocol to extract the protein from the gel?

Dear Fernanda,

We had purified protein from the gel for amino acid sequencing as following: The protein bands in the SDS slab gel were cut out, and soaked in the buffer containing 20mM Tris and 1% SDS（pH8.0） for 2 days at room temperature. After extract of containing proteins were filerred to purge fragments of gel, the constituent proteins were precipitated by trichliroacetic acid precipitation.

However, sample collection rate may be low.

Hope this might help.

Thank you Takao Kuwada, I'll try to do this!!

You can purify your protein from SDS-PAGE gels by electroelution.

For sequencing, we always transfer to immobylon membranes, stain them with red Ponceau and excise the band.

Hi, I've extracted protein from native PAGE simply by crushing the gel through syringe few times and then overnight incubation in buffer. I think that should work also for SDS-PAGE.

Electroelution is the best. You do not need an instrument, you acn do it in a dialysis bag.

Hi Fernanda.

There is another, more popular approach, if this is a natural protein and you have facilities for MS/MS. Cut off band of PAG containing protein and go to mass-spectrometer people. Many of them know how identify protein or even sequence it.

Good luck,

Yuri

Hi Fernanda,

Why do you not want to blot the protein onto sequencing grade PVDF, stain with coomassie blue, excise and then submit for Edman sequencing? This is a very straight forward method and it works very well.

Karen

Hi Karen, sometimes this method doesn't work well here, I'm looking for another method that avoids this protocol.

Fernanda, there's a kit from proteabio which successfully elutes proteins from cut out bands in Coomassie stained SDS-PAGE. They also offer reasonable pricing on the whole MS screen including extraction. All you would need to do is sent them your cut out band. They're really great. www.proteabio.com

Good luck! Bettina

SDS-PAGE extracts contain a lot of garbage that interferes with Edman degradation. Applied Biosystems used to offer the ProBlot membrane system to purify the protein from the extract. Alternatively you could pass the extract over a small RP-column or just a RP-precolumn and and after washing with 0.1 % TFA, elute the protein with acetonitrile from that material. Unfortunately membrane proteins usually do not come off from RP-material.

Dear Fernanda

If your purpuse is just to extract and identify your protein there is reliable and LCMSMS compatible protocol using trypsin digestion that can do that for you. But if you are interessed to extract intact proteins for full sequencing purpuses this protocol may not be appropriate. Do your target protein show a single band in your gel? if yes your protein can go for seqencing without any problem. if not you may need to think about further purification steps like HPLC. For Edman degradation seqencing, samples are usualy required to be realy pure. hope this can help!

Regards Ibtissam

Hi, my protein shows a contaminant in the SDS gel, so I want to do the Edman's degradation to know what is this contaminant. In LCMSMS is very difficult to find the n-terminal. I tried this protocol with trypsin, but without sucess. I think for to eliminate this contaminant I will have to do another step of reverse phase... but the acetronile makes my protein to lose it activity. So, I have an impass here! =/

Hi

2D electrophoresis will help sometimes to remove the contaminated protein band