Ni-IDA Sepharose gel for His Tag Protein Purification

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Description

Biotool Ni-IDA Sepharose gel for His Tag Protein Purification uses sepharose CL4B as the substrate. And Ni metal ion is chelated onto the gel through the classical IDA ligand. Ni-IDA ligand has a stronger binding ability to His-tag in which affinity reaction every Ni2+ and Histone amino acid forms two coordinate bond. Biotool Ni-IDA Sepharose gel for His Tag Protein Purification acquires binding capacity of 40 mg/ml at most, and with its nature of stability, simplicity, good repeatablity, and convenience, Biotool Ni-IDA Sepharose gel is an ideal tool for your demand of His-tag recombinant protein purification.

Storage

Biotool Ni-IDA Sepharose gel for His Tag Protein Purification is provided as 50% slurry and is suggested to be stored at 4℃.

Protocol

His-Tag Protein Purification

Lysis buffer: 20-50 mM Tris-HCL, 0.25-0.5M NaCL, pH7.3-8.3. It is suggested to add protease inhibitor (like cocktails) to protect your target protein from degradation. And 5%-10% glycerol in lysis buffer will also provide protection. 2mM 2-ME can be used as reducing reagent. EDTA and DTT should be avoided in the lysis buffer which will destroy the normal function of Ni ion in the Sepharose gel. 
Equilibrium buffer: 20-50 mM Tris-HCL, 0.25-0.5M NaCL, pH7.3-8.3. 
Washing buffer: 20-50 mM Tris-HCL, 0.25-0.5M NaCL, pH7.3-8.3, 10-20 mM imidazole. 
Elution buffer: 20-50 mM Tris-HCL, 0.25-0.5M NaCL, pH7.3-8.3, 250 mM imidazole. 
1. Collect the 500mL culture media of E.coli expressing your target recombinant His-tag protein by cetrifugation. Cells are resuspended in lysis buffer and treated by ultrasonic or freeze-and-thaw. Centrifuge and collect the supernatant. 
2. Load the sepharose gel to a gravity column. Balancing with 10 times the column volume of Equilibrium buffer by natural flow. 
3. Mix the supernatant with balanced sepharose gel. And bind for 1-1.5h at 4℃ by low speed rotation. 
Note: the binding tube should be filled up with lysis buffer and the foam on the liquid surface should be removed before rotation, then foam generation during rotation will be eliminated which is unfavourable for protein binding. 
4. Load the sepharose gel binding mixture to the gravity column, collect the flow through by natural flow. Wash the sepharose gel by 10-20 column volume washing buffer until the OD280 of effluent is less than 0.02. 
Note: for protein which is purified for the first time, the imidazole concentration should be tested and effluent samples should be kept for further analysis. For example, if large quantity of target protein appears in the washing effluent, the imidazole concentration should decrease to 5 mM or lower, if the amount of background protein is large, the imidazole concentration should increase and larger volume of washing buffer should be used. 
5. Add 4-10 times column volume elution buffer onto the sepharose gel and collect elution. Usually the target His-tag protein will appear in the second volume of elution buffer. For some special proteins, elution could be achieved by EDTA pr pH gradient. 
6. After elution, the sepharose gel should be rinsed with 10 times column volume 1% acetic acid, be balanced with neutral pH TBS, be rinsed with large amount pure water, and be stored in 20% ethanol. Store the sepharose gel in sealed tube at 4℃, do not freeze at -20℃, nor dry the gel.

Regeneration of Ni-IDA Sepharose gel for His-Tag Protein Purification

Ni sepharose gel could be used repeatedly and present the same purification efficiency. When the original column is applied for a different target protein, or the Ni metal ion drop off after long usage, and the Ni resin column shows blockage and low flowing speed, the Ni resin column cloud be regenerated as the following method: 
1. Wash the resin with 4 times column volume 0.1M EDTA solution to thoroughly wipe off Ni metal ion, and rinse thoroughly with 10 times column volume distilled water to eliminate EDTA residue. If the resin column is seriously clogged, incubate within 1% Triton X-100/TBS solution overnight. 
2. Wash the resin column with 4 times column volume 0.1M HCL, 10 times column volume distilled water, and then 4 times column volume 0.1M NaOH, balance with TBS of neutral pH, and then wash with large amount of water. In this step, long immersion in strong acid or strong base solution should be avoided. 
3. Incubate the resin with 4% nickel dichloride or nickel sulfate solution for 10 minutes to achieve re-chelate. 
4. Add proper amount of 20% ethanol. Store the resin in sealed tube at 4℃, do not freeze at -20℃, nor dry the gel.

Supplemental Information:

1. When the stability of target His-tag recombinant protein is unknown, the whole purification process should be manipulated at 4 ℃. Protease inhibitor could be added to protect target protein from degradation, and EDTA should be added after purification. And certain nonionic detergent could be added to increase binding efficiency, including 1% Triton X-100, 1% Tween-20 or 0.1% NP-40. 
2. Its better to use separate resin column for different protein purification. 1 ml of Ni-IDA sepharose gel could bind 20-40 mg His-tag protein, if the amount of target protein is more, larger volume of resin could be used. 
3. 1 mM imidazole could be added into lysis buffer to reduce binding of background protein. Some target protein will elute in washing buffer containing 20 mM imidazole, so it is necessary to keep every effluent sample for SDS-PAGE analysis and search for the proper experimental parameter. 
4. If the target protein is expressed in the form of inclusion body, it is usually to add 8M urea and other detergent to release the protein in denaturation. The denatured sample should be treated properly, or the resin column will be clogged. 
5. Besides the normal imidazole elution, EDTA and pH value gradient could also be used to elute protein, but which two have obvious disadvantage and can only be used in special situation. 6. The His-tag in the recombinant could be wiped off by enzyme digestion. TEV protease is suggested which has better specificity.

Trouble Shooting

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