Which experiment could further identify an unknown protein besides SDS-PAGE and Western Blot? How implement the results obtained from these two methods?
If you performed a Western blot, you must already have some information about the identity of the protein, since you would have had to use an antibody that recognizes it.
Aside from that, the amino acid sequence should be determined. The protein band can be cut from the gel and sent to a specialized protein mass spectrometry lab for sequencing. By comparison of the sequence with the proteome of the organism from which the protein was extracted, its identity can often be found.
Neither SDS-PAGE, nor Western blot identify any unknown proteins (also see Adam's comment).
To identify protein, you have to do MS analysis of purified protein. For purification you can use, besides others, also electrophoretic methods as SDS-PAGE, native PAGE, 2D electrophoresis, isoelectric focusing.
Depending how “unknown” your protein is LC-MS might not be good enough. You might need to find a lab that does de-novo protein sequencing, not trivial these days
Respected Yoram sir, De- novo protein sequencing is used for only to determine how many of each kind of amino acids are present in purified protein.
MS with ESI (Electro spray ionisation) under MALDI-TOFF (Matrix assisted laser desorption ionization time of flight) is best way for separartion, purification and quantitivation of protein.
Respected Tole S.B. I beg to differ. Been there tried it, and still stuck with my unknown protein
MALDI-TOFF-MS is a great tool to identify protein that resemble known proteins sequence (by comparison to library), but if you have no reference now what?
If you know something I don't I will love to learn!
And to know "how many of each kind of amino acids are present in purified protein" you do "amino acid analysis", much simpler analysis
Respected Yoram sir,
I thing sir MALDI-TOFF is suitable for all types of protein for identification.
"how many of each kind of amino acids are present in purified protein" this is done by using alkylation of amino acids (Sangers dideoxy method i.e.N- terminal sequencing ) and also by Edman degradation.
These two methods are very suitable for determination of no. of amino acids in purified proteins
Yoram Gerchman What organism do you work with? Is there no genome information, even for anything related?
MALDI-TOF should give you peptide sequences. You can always try to BLAST it to get at least some idea. And using the peptide sequences, you can design (degenerated) primers to clone your GOI.
Thanks @Tomáš Hluska, we are working on the myrosynase enzyme from Ochradenus baccatus (https://flora.org.il/en/plants/OCHBAC/) a desert plant. It is in the same Order as Arabidopsis thaliana (Brassicales)but rather remote. To the best of my knowledge no genome data :-(
In addition to SDS-PAGE and Western blot, there are several other experimental methods that can be used to further identify an unknown protein. Some of these techniques include:
Regarding the implementation of results obtained from SDS-PAGE and Western blot, these techniques provide valuable information about the molecular weight and presence/abundance of the protein, respectively. They can help confirm the presence of the protein, assess its size or modifications, and provide an initial indication of its expression level. The results obtained can guide further experiments using the techniques mentioned above to obtain more detailed information about the protein's identity, structure, function, and interactions.
It's important to note that the choice of additional experiments depends on the specific goals and characteristics of the unknown protein. Each technique has its strengths and limitations, and a combination of approaches is often necessary to fully characterize and identify an unknown protein.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
Thanks, Nikolay Klyashtornyy this is a rather conclusive list and thus less useful :-(
- Badges
- Science topic