Protein purification depends upon the properties of the protein. It will also depend on what equipment you have (Ultra-centrifuge, FPLC or gravity column, what types of resin, do you have dialysis supplies, what protein quantification assays do you have, etc). For Hexahistidine tagged purification I've had good luck with Nickel-NTA or Nickle-Sepharose followed by a cleanup with MonoQ.

If you're using an FPLC, I'd recommend you get advice before even touching the machine. They tend to be fickle, and it takes a while to learn how to safely use them. One wrong setting can destroy a purification.

For a gravity column (less expensive), Qiagen's manual for Ni-NTA is actually a pretty good place to start.

https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en

Dear Sucaad

for protein expression there are many protocolls and media available. My prefered media is Enpresso (http://enpresso.de/en/) which is an high density media that allow to obtain from 75ml of culture the same biomass that you can obtain with 500ml of LB with comparable specific expression and solubility level. In this way, if you have to express few proteins you can avoid small scale expression trials that we normaly do with LB but you cn go directly to 75ml scale and in case of positive results proceed directly with the putification of your biomass.

Attached you can find the protocoll that i'm using for protein expression.

To check expression and solubility you can collect a small culture amount before and after induction and process it following the protocol

Method Multiwell based protein expression and solubility test

Afterwards if your protein is expressed and soluble you can proceed with purification of the entire biomass stored at -20°C using IMAC chromatography.

The amount of resin t be used it depends form the expression level of your proteins. From a single flask 75ml you will obtain 2-3g of wet biomass that can produce 1-10mg of recombinant protein depending from the expression level, therefore you can use from 300 to 2ml of resin in a appropriate gravity flow coloumn.

Empty GE pd-10 for example are working well fro resin volume of 1 - 2ml. For lower resin volume (200-500ul) you can use of the Biorad Biospin disposable coloumns.

Use a limiting resin volume can result in lost of some protein but in gihg purity because you saturate the resin with your protein.

For 6His tag generally i'm using a lysis/binding buffer with 20mM Tris, 300mM Nacl, 10mM imidazole pH=8 and a

Wash with 20mM imidazole

and elution with 300mM imidazole.

If you do not have a sonicator, you can use CelLytic express of Sigma ad lysis agent and just dilute 1:3 your soluble fration in the tris,nacl,imidazole binding buffer before IMAC.

For 10 His tag you can wash with 30 mM and also 50mM sometimes with no detach of your protein.

Of course for expression you can also use LB. In this case i suggest to you to growth bacteria at 37°C induce expression at =D 0,6-0,8 and incubate bacteria 5h at 25°C that generally represent a good compromise in terms of solubility and expression. Afterwards if you are not safistied of expression or solubility level you can reperat the test at higher temperature (if you have low expression and good solubility) or low temperature (if you have good expression and partial solubility).

good luck

Manuele