purify unknown protein

[shade]

dear friends:

i'm working on unknown protein. i've done the antimicrobial assay and it showed positive result. now i want to purify my unknown protein but I'm blank what to do 1st and the procedure to follow.

how do i arrange these things in order to solve my problem:
extraction
SDS-PAGE
RP-HPLC
MALDI-TOF
IEC

which one is 1st and next...please help. i'm a student.

[danfive]

I do this all the time. If you have a simple band and can cut it from your gel using aseptic technique, you can do a SDS-PAGE, stain with MS-compatible stain (Imperial Stain from Pierce) cut your band of interest out, put in clean 1.5ml Epi tube. Then perform the typical in-gel digestion (simple, buy a kit, here's a protocol ---> [Only registered users see links. ]). Now you have trypsinized peptides ready for MALDI-TOF. Good hits on the MALDI and you will be able to identify your unknown protein.

If that doesn't satisfy you, perform the HPLC and IEC before MALDI-TOF.

[shade]

dear danfive:
thanks for the information. i'm so glad that u care to share with me and make my life easier. tQ. i'll try it A.S.A.P!

[danfive]

Here is some info, in case you need to try the LC and IEC. #1 is the best for protein stuff, but they are all useful.

Originally Posted by danfive

Here are some powerpoints with useful liquid chromatography intros, summaries, tips
1. "Protein Chromatography" [Only registered users see links. ]

2. "Advanced Chromatographic Methods" [Only registered users see links. ]

3. Good Primer on Chromatography "What is Chromatography?" [Only registered users see links. ]

[shade]

i'm working on my experiment...tQ danfive!

[MatthewDooley]

I think that you have more knowledge about this protein purifier. I want to say thanks because here you have shared your knowledge with us.

[shade]

do you have any protocol for methanol extraction for microbe?i diluted my lyophilized sample using PBS but it didn't diluted well. i have a very limited sample. so i'm so thankful if you all have any suggestion. at the end i want to fractionate my sample and do antimicrobial test.

[MatthewDooley]

As I have visit this post and come to know that you have much knowledge of protein so that I want to ask you that can we take expired [Only registered users see links. ]? I mean if we take it than is there any side effect of this expired protein powder?

[danfive]

Originally Posted by MatthewDooley

As I have visit this post and come to know that you have much knowledge of protein so that I want to ask you that can we take expired [Only registered users see links. ]? I mean if we take it than is there any side effect of this expired protein powder?

LOL, this is a food product (its been cooked/pasteurized, dried, etc), so like most you can use them after the expiration date. Go by smell & taste as those are the main reason why expiration dates are on there.

If you want some real expert opinion try asking bodybuilders, like here ---> http://forum.scoobysworkshop.com/

[MatthewDooley]

Originally Posted by shade

dear friends:

i'm working on unknown protein. i've done the antimicrobial assay and it showed positive result. now i want to purify my unknown protein but I'm blank what to do 1st and the procedure to follow.

how do i arrange these things in order to solve my problem:
extraction
SDS-PAGE
RP-HPLC
MALDI-TOF
IEC

which one is 1st and next...please help. i'm a student.

According to me it is impossible........