How can I purify the wanted protein - (May/21/2007 )
Good afternoon!
I'm already succesful in cloning and expression interested gene in pHCE vector in BL21 E.coli. 
After disrupting the harvested cell, I check by SDS-PAGE and it's lucky 'cause of tagged protein in Soluble form. But I faced with a big problem when purification these protein from other unwanted protein because of pHCE vector no have any agent to purify!!! 
How can I purification wanted Protein??? please give me some your advices and experiences!!!
Plz answer me as soon as possible because I must report my experiment result at the end of this month. I very worry!
Thanks for your helps!!! 
I'm already succesful in cloning and expression interested gene in pHCE vector in BL21 E.coli.
After disrupting the harvested cell, I check by SDS-PAGE and it's lucky 'cause of tagged protein in Soluble form. But I faced with a big problem when purification these protein from other unwanted protein because of pHCE vector no have any agent to purify!!! How can I purification wanted Protein??? please give me some your advices and experiences!!!
Plz answer me as soon as possible because I must report my experiment result at the end of this month. I very worry!
Thanks for your helps!!!
Hi!
If you have no tagg so only common chromatographic techniques should use! Do you know pI of your protein? The first one method to begin enrichment is IEC. If you don't know pI make pilot exp on DEAE and SP sepharose at different pH. Analyse binding by SDS PAAG and choose optimal pH range and resin. If you are in a hurry try to dialyse your protein mixture against 20mM NaP pH 7.4 and apply on packed cation or anion exchanger then elute in slight Salt gradient till 1M NaCl. May be you will be luck and your protein have retention on column and not fall through.
Also the other variant RP resin C18 or C8 for the crude extract enrichment use resin in low pressure system and elute with ACCN gradient.
After first enrichment you should analyse purity of your protein on SDSPAAG , then you should decide to make GF ( if impurities ( accord your Mw) are far enough from your targeted protein ( resin - depends on your protein Mw, write and we will discuss ) or if impurities are near by Mw your protein it will be better to use hydrofobic chromatography or FPLC\HPLC IEC to enhance effectiveness of purification.
Do you have AB against your protein. Is it possible for you to prepare immunoaffinity column. it will be a good choice!
It is first tips other aspects we will discuss
Could you please tell the name of your protein?
if it is not a novel protein or identical to known protein, you can find a protocol for it in internet.
If it a novel protein without identical protein, you start the process by NH4SO4 precitpiation, Ion chromatography, Filtration, vv, but it needs experiences.
Good evening!
Thank you for you answers. But I very sorry because my knowledge about your method very little and I don't have AB against my protein 
But for you know more about my work and easy discussing I will tell details about my experiment
The aim of my experiment is preparing recombinant protein as antigen source for making antibody, so in my opinion it isn't very important if protein is native or denature.
steps in my experiment fellow: BL21 E.coli containing pHCE-VP19 recombinant plasmid ( VP19 is envelope protein of virus) was cultured in LB medium (containing 100µg/ml) at 37°C over night. The harvested cell was resuspended in TE buffer (50mMTris-Cl,, 2mM EDTA, pH8.0) and sonicated until the liquid becomes clear, then centrifuged for separating soluble and insoluble fractions and then run SDS-PAGE, then dye gel by comassie blue. After confirming the wanted band ( 19kDa) I cut it out and mice it + vortex in Tris-Cl/ 1%SDS, stand it overnight at room temperature, then this sample were centrifuged, the VP19 protein were obtained. The next step I must determine concentrate of this protein for injection into rabbit. But I can’t determine because of blue color in my elution protein. So how can I separate protein from comassie blue???
So plz tell me about my opinion and what’s wrong in my experiment????
Plz recommend to me some better methods with protocol fellow.
Thank you for your help!
Thank you for you answers. But I very sorry because my knowledge about your method very little and I don't have AB against my protein
But for you know more about my work and easy discussing I will tell details about my experiment
The aim of my experiment is preparing recombinant protein as antigen source for making antibody, so in my opinion it isn't very important if protein is native or denature.
steps in my experiment fellow: BL21 E.coli containing pHCE-VP19 recombinant plasmid ( VP19 is envelope protein of virus) was cultured in LB medium (containing 100µg/ml) at 37°C over night. The harvested cell was resuspended in TE buffer (50mMTris-Cl,, 2mM EDTA, pH8.0) and sonicated until the liquid becomes clear, then centrifuged for separating soluble and insoluble fractions and then run SDS-PAGE, then dye gel by comassie blue. After confirming the wanted band ( 19kDa) I cut it out and mice it + vortex in Tris-Cl/ 1%SDS, stand it overnight at room temperature, then this sample were centrifuged, the VP19 protein were obtained. The next step I must determine concentrate of this protein for injection into rabbit. But I can’t determine because of blue color in my elution protein. So how can I separate protein from comassie blue???
So plz tell me about my opinion and what’s wrong in my experiment????
Plz recommend to me some better methods with protocol fellow.
Thank you for your help!
There are 2 methods:
1. You run your crude extract in 10 lanes, and then cut 1 lane for staining. After staining, u can know its position, then cut the remaining 9 lanes of your protein (that contains no dye).
2. You can estimate the quantity of your protein by comparing the density of a series of BSA in the SDS-PAGE.
Hope it help!
The aim of my experiment is preparing recombinant protein as antigen source for making antibody, so in my opinion it isn't very important if protein is native or denature.
Not really. If antibodies recognize denatured proteins, they may be good for detection of those (e.g. in WB), but may not recognize natural proteins (i.e. in other exps with living cells,... when the antigen is still fold naturally. So your Ab may work for WB but not IF, IP, FACS.... It all depends on epitopes.
There is a kit from Pierce to calculate protein concentration when the samples have been incubated with dyes or boiled in commassie blue. It will take a number of steps, but it does work. Otherwise, you can compare sample with parallel runs of diffrent amount of control proteins (like BSA, GST...)
Hope it help!
Concerning Almasy second reply I did it also and you can estimate approximately protein concentration by this techniq
Hi
I always use BSA to estimate the protein concentration of my target protein on a Coomassie stained gel. I usually load 5, 2.5, 1.25 ug BSA, scan in the gel and analyse the image by AlphaEaseFC software. I haven`t had any problems with this approach so far.
Good luck
TCA precipitation and washing with methanol should destain proteins from CBB
Sometimes TCA doesn't work properly! It depends on concentration level of your protein and buffer solution where your protein was dissolved. I 've had some difficults using TCA for some proteins.