This protocol should work well for your 37 kDa protein, assuming it's expressed with a protin tagged with a polyhistidine tag (His-tag), typically consisting of 6 histidine residues.

Materials Needed :

- Ni-NTA Econo column or similar resin (available commercially, e.g., from Bio-Rad, Qiagen, etc.)

- Binding buffer:Typically, 50 mM sodium phosphate, 300 mM NaCl, pH 7.4, with 20-30 mM imidazole (this prevents non-specific binding). - Washing buffer:Same as the binding buffer but with 50-100 mM imidazole. - Elution buffer:Same as the binding buffer but with 250-500 mM imidazole to competitively elute the His-tagged protein. Take imidazole with the highest possible purity this will limit the absorbance to 280nm during the elution gradient. - Cleared bacterial lysate (containing the His-tagged protein).

- Protease inhibitors (optional, to protect protein from degradation).

- Centrifuge (to pellet bacterial cells).

- Buffers for protein analysis (e.g., SDS-PAGE, Bradford for protein quantification).

Harvesting the Bacterial Culture

- Grow the bacterial culture (e.g., E. coli) in appropriate medium (e.g., LB or minimal media) at 37°C with the required inducer (e.g., IPTG) to express the His-tagged protein.

- When the culture reaches the desired optical density (OD600, typically 0.6-0.8), induce protein expression.

- After induction, grow the culture for an additional 2-4 hours (depending on your protein's expression time).

- Pellet the bacteria by centrifugation at 4,000–6,000 x g for 10 minutes at 4°C.

Cell Lysis and Lysate Preparation

- Resuspend the bacterial pellet in a suitable lysis buffer (e.g., 50 mM sodium phosphate, 300 mM NaCl, pH 7.4), adding protease inhibitors if necessary.

- Lyse the cells using a method such as sonication, French press, or chemical lysis. The choice of method depends on the solubility and characteristics of the protein.

- Centrifuge the lysate at 12,000–15,000 x g for 20–30 minutes at 4°C to remove cell debris.

- Collect the supernatant, which contains the soluble His-tagged protein.

Ni-NTA Column Preparation

- Equilibrate the Ni-NTA resin (Econo column or other) with at least 5-10 column volumes (CVs) of binding buffer (e.g., 50 mM sodium phosphate, 300 mM NaCl, pH 7.4).

- If using a gravity flow column, make sure to pre-rinse the column with binding buffer until the flow rate stabilizes.

Protein Binding to the Column

- Load the cleared lysate (preferably filtered through a 0.45 µm filter) onto the Ni-NTA column.

- Allow the lysate to pass through the column by gravity flow or gentle pump flow.

- Let the resin bind the His-tagged protein for 5-10 minutes to ensure maximum binding. You can gently agitate the column if needed.

Washing the Column

Wash the column with 5-10 CV of washing buffer (binding buffer with 50-100 mM imidazole). This helps to remove non-specifically bound proteins.

Monitor the flow-through to check for any contaminating proteins.

Elution of His-tagged Protein

- Elute the His-tagged protein by applying elution buffer (binding buffer with 250-500 mM imidazole).

- Collect fractions (typically in 1-2 mL aliquots), starting from the first elution and continue until the protein is completely eluted.

- You may choose to gradually increase imidazole concentrations for a more refined elution gradient, depending on your protein’s affinity and purity.

Analysis of Eluted Fractions

- Analyze the elution fractions by SDS-PAGE and Coomassie staining or western blotting using an anti-His antibody, if required.

- Determine the fraction with the highest protein concentration (eluted protein).

Protein Concentration and Buffer Exchange (if necessary)

- If needed, concentrate the protein using a centrifugal concentrator (e.g., Amicon Ultra) and exchange it into an appropriate buffer for downstream applications (e.g., storage buffer, enzymatic assays, etc.).

- For buffer exchange, you can use dialysis membrane or desalting columns.

Hello! I used the kit handbook from QIAgen called "The QIAexpressionist". The buffers are important and the washes. It has a protocol for the spin columns which may be more what you need. Have a look at this handbook. If the link doesn't come up just google "QIAexpressionist" Good luck! https://www.qiagen.com/us/resources/resourcedetail?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en

Dear Abdul Rouf

you can find some hints aobut imac purification on

https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html

https://www.blogger.com/video.g?token=AD6v5dzP9DkeYt4nwIFl1pNVQuJw7_vnHlvCcbCTzad5M6URrbtpOEJ9Z5MJ-gTcfR6n06A5fXwQmD5qJtKayesvHrQtqzfE0mhrKxtPpHHuJO0u2FMq9kmvLSz60LTG_RLcHgTUWkY

as Matthew Edward Thornton suggest toy the qia expressionist is even a good way to be introduced to IMAC

best

Manuele

First, you need to determine the amount of your target protein. This will help you choose an appropriate purification protocol. Spin columns are a simple and convenient option but are only suitable for small-scale purification. For larger quantities, you can connect your columns to an ÄKTA or Bio-Rad purification system to scale up production efficiently.