protein purification

[magnificientleo]

hi
i hv to purify a recombinant fusion protein which is having his tag.i used ni column but when i am running eluted fractions in 12% sds page ,i am not getting a single band..instead i am getting 3 bands in almost all the eluted lanes ...can anyone help me with this??

[moleculardude]

Hey there Leo,
welcome to the forums buddy.

The bands you are getting are you sure they are not your blocking agent ie BSA or some other proteins?

Also, how are you eluting? Maybe the eluting steps are very close to the same... are you increasing stepwise salt / buffer pH conditions?

I would check carefully how you are eluting and how you visualize those eluted proteins (are you using coomasie?).

[tuttu]

Hi,

I am not sure about the way u use the columns, i generally use a freshly packed and a properly pretreated column for final elution and run through the elute into another similar column and re-elute. This has helped me a couple of times. It wud easier to figure out wat exactly the problem is if u cud elaborate more.

good luck!!

[MicroKiller]

A more detailed description would help.. but i would suggest that you run your load and wash fractions on the gel to .. jst to see if you have the same three bands... cause this could be an overload of a common protein you have in the fraction which is sticking to the ni columns. Did you regenerate your ni columns and etc... check your binding and elution buffers to be on the safe side...

best of luck

[ruby]

how are u eluting ur protein? u need to run ur crude sample along with ur eluants, it wud tell u whether its there from begining, or getting degraded???

you can do mALDI to confirm its identity,

[sergbio]

Hi!
First of all this affinity step is only step for enrichment of your protein from mixture but in many cases is not a final step of purification procedure.

I will try to make some recomends to better enrichment of your protein on Ni NTA

What kind of elution did you apply? Ph dependent or imidazole gradient?
Most likely it is nonspecific interactions! If your protein is from inclusion bodies, try to purify your protein under denaturated conditions Bind it on Ni column in 6M Gua-Hcl Tris 20mM 1mM betamercaptoethanol 5mM imidazole pH 8.0. Then wash column 5 volumes of column, then wash 10volumes of buffer 2( 6M urea 1mM bme, 20mM imidazole pH 8) Try to make step elution for example 40mM 3-4 column volume then 70 mM and so. When the peak appears on one of these steps elute it till baseline at this imidazole concentration, then rise imidazole conc


Sometimes it is possible to get good purification by washing column before imidazole elution at PH near pKa of His residue. So only strongly multiple binded proteins will return on resin. But of course yor risk to wash your protein in some extent.
May be there are other contominant proteins which contain multiple His residues. So It is difficult to purify on this step. Try to apply ion-exchange or SEC chromatography ( in low pressure chromatography or HPLC ).

[sergbio]

Hi!

Here is some PR concerning NI NTA purif of His proteins



About cleaning NI NTA column

After elution, the column can be cleaned by washing with 0.2 M acetic acid/6 M guanidineHCl, then reequilibrated with IN column buffer B. Under the conditions described, the column can be used two or three times before it turns from light blue-green to brownish-gray, indicating that regeneration is required. The column can be regenerated by passing 2 column volumes of 100 mM EDTA to strip Ni from the resin, washing the column sequentially with 2 column volumes each of 0.1 M NaOH and 6 M guanidineHCl, and finally passing 2 column volumes of 10 mM NiSO4 through the column to charge the resin with Ni. The column is then washed with water and equilibrated with IN column buffer B.

IN column buffer B

To 500 ml H2O add:
10 ml 1 M TrisCl, pH 8.0 (pH as determined at 4C; 10 mM final)
0.73 g NaH2PO4H2O (monobasic; mol. wt. 137.99)
13.44 g Na2HPO4 (dibasic; mol. wt. 141.96)
0.07 ml 2-mercaptoethanol (2-ME; 1 mM final)
After salts have dissolved add:
573.2 g guanidineHCl (6 M final)
Bring temperature to 4C and add 4C H2O to 1 liter
Adjust pH to 8.0 (as determined at 4C) if necessary with 2 M NaOH or 2 M HCl
Prepare immediately before use





NATIVE MCAC FOR PURIFICATION OF SOLUBLE HISTIDINE-TAIL FUSION PROTEINS


1. If extract is frozen, thaw on ice. Load onto Ni2+-NTA column and allow to flow through at a rate of 10 to 15 ml/hr. Collect column flowthrough and save for SDS-PAGE (see Support Protocol 1).

Charged NTA resin has a capacity of 5 to 10 mg histidine-tagged protein per milliliter of packed resin. The amount of extract that can be loaded on the column will depend on the amount of soluble histidine-tagged protein in the extract.

2. Wash column with 5 ml MCAC-0 buffer at a flow rate of 20 to 30 ml/hr. Discard flowthrough.

3. Wash column in stepwise fashion with 5 ml each of MCAC-20, MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-200, and MCAC-1000 buffers at a flow rate of 10 to 15 ml/hr. Collect 0.5-ml fractions and save on ice for SDS-PAGE

Alternatively, the column can be eluted with a 5-ml linear gradient of 0 to 400 mM imidazole in MCAC buffer.

The second and third fractions of each wash will contain most of the eluted proteins. Most proteins with hexahistidine tails will remain bound in 60 mM imidazole (MCAC-60) and elute with 100 to 200 mM imidazole (MCAC-100 or -200); therefore, the purified protein will elute in MCAC-100 or -200. Proteins with longer histidine tails (e.g., 10 residues) bind to Ni2+-NTA with greater affinity and require higher imidazole concentrations for elution. However, optimum washing and elution conditions must be determined for each protein.

Once optimum washing and elution conditions are established, it is possible to prepare the crude extract in a buffer that contains the highest imidazole concentration in which the histidine tail remains bound to the Ni2+-NTA (e.g., MCAC-40 or -60 buffer). This decreases nonspecific binding of proteins to resin and permits use of a single buffer for extract preparation, column loading, and column washing.

4. Elute column with 1 ml MCAC-EDTA buffer at a flow rate of 10 to 15 ml/hr, collecting 0.5-ml fractions.

The blue-green color of the column will disappear as nickel is removed by EDTA. More tightly bound proteins may be found in these fractions. The resin can now be recharged (repeat steps 6 to 8) and the column reused.

If protein is eluted adequately with imidazole (as determined by overall yield for subsequent preparations), EDTA washing can be omitted. The column can be reequilibrated with MCAC-0 buffer and the purification repeated. The same column can be used three to five times before EDTA stripping and nickel recharging are necessary. Only one protein should be purified on any given column.

5. Analyze fractions for the presence of eluted protein.

An ultraviolet (280-nm) absorbance flow monitor is helpful for following column elution but is not necessary. An alternative is to measure the OD280 of individual fractions to identify protein-containing fractions. However, imidazole will also absorb at 280 nm.

A quick and easy method to determine which fractions contain eluted protein is to place 2 ul undiluted Protein Assay Dye Reagent Concentrate (Bio-Rad) on a piece of Parafilm, add 8 ul from fraction to be tested, and mix by pipetting up and down. Immediate appearance of blue color indicates that the fraction contains protein. This does not work in the presence of Triton X-100 because the detergent itself produces an intense blue color; for this reason, Triton X-100 is excluded from the washing and elution buffers.

6. Combine the fractions containing eluted protein and remove a 10-ul aliquot for SDS-PAGE (see Support Protocol 1). Freeze the remainder in smaller aliquots at 70C or in liquid nitrogen.

If a different buffer for the protein is desired (e.g., for proteolytic removal of the histidine tail), the protein should be dialyzed against the buffer of choice to remove the MCAC buffer prior to storage at 70C or in liquid nitrogen.

7. If time permits, proceed immediately to analysis of fractions by SDS-PAGE and processing of protein (see Support Protocol 1). Otherwise, freeze all samples at 70C until ready for analysis and processing.

MCAC buffers (store 6 months at 4C)

MCAC-0 buffer:

20 mM TrisCl, pH 7.9
0.5 M NaCl
10% glycerol
1 mM PMSF (phenylmethylsulfonyl fluoride)

MCAC-1000 buffer:

20 mM TrisCl, pH 7.9
0.5 M NaCl
10% glycerol
1 M imidazole
1 mM PMSF

Add PMSF immediately before use from a 0.2 M stock in 100% ethanol stored at room temperature. All buffers used with Ni2+-NTA resin contain high salt concentrations to reduce nonspecific electrostatic interactions between proteins and resin. Lower salt concentrations can be used but may lead to nonspecific binding of unwanted proteins to the resin.

MCAC-20, MCAC-40, MCAC-60, MCAC-80, MCAC-100, and
MCAC-200 buffers:

These buffers (containing different concentrations of imidazole) are made by mixing MCAC-0 buffer and MCAC-1000 buffer in the appropriate ratios: e.g., for MCAC-60 buffer, 94:6 (v/v) MCAC-0/MCAC-1000.







DENATURING MCAC FOR PURIFICATION OF INSOLUBLE HISTIDINE-TAIL FUSION PROTEIN



Prepare cell extract

4. Thaw cell pellet (from step 1) on ice. Resuspend in 5 ml GuMCAC-0 buffer by pipetting, sonication, or homogenization.

5. Freeze 10 min at 70C and thaw at room temperature.

Protease inhibitors are omitted because proteases are inactivated by guanidine. Triton X-100 is not needed at this step. Freezing is not necessary but is included because it ensures complete lysis of cells.

Subsequent steps can be performed at room temperature. However, if solid-phase renaturation is used (see Alternate Protocol 2), it is better to maintain lower temperatures throughout the process.

6. Gently mix samples for 30 min using a rocker, rotating mixer, or magnetic stirrer. Centrifuge 15 min at 27,000  g (15,000 rpm in Beckman JA-20 rotor), 4C. Decant supernatant into a clean container and discard pellet. Set aside a 10-ul aliquot for analysis by SDS-PAGE (see Support Protocol 1).

The supernatant can be frozen at 70C indefinitely before continuing with the procedure.

Purify protein

7. If extract from step 5 is frozen, thaw at room temperature. Load onto Ni2+-NTA column and allow to flow through at a rate of 10 to 15 ml/hr. Collect flowthrough and save a 10-ul aliquot for SDS-PAGE (see Support Protocol 1).

8. Wash column with 5 ml GuMCAC-0 buffer at a rate of 20 to 30 ml/hr. Discard the flowthrough.

9. Wash column in stepwise fashion with 5 ml GuMCAC-20, -40, -60, -100, and -500 buffers at a rate of 10 to 15 ml/hr. Collect 0.5-ml fractions and save for SDS-PAGE (see Support Protocol 1).

The second and third fractions from each wash will contain most of the unbound protein. The histidine tail binds slightly less avidly under denaturing conditions. Lower imidazole concentrations are therefore required for washing and elution than in the Basic Protocol.

10. Elute with 1 ml GuMCAC-EDTA buffer at a rate of 10 to 15 ml/hr, collecting 0.5-ml fractions.

11. Identify fractions containing the protein, pool together, transfer to dialysis tubing, and seal.

Alternatively, fractions can be frozen at 70C indefinitely before continuing with the procedure.

Guanidine precipitates in the presence of SDS and must be removed by dialysis before SDS-PAGE. An alternative technique employs buffers that switch from 6 M guanidine to 8 M urea during affinity column washing (Stuber et al., 1990). This permits samples to be taken directly from urea fractions without dialysis and analyzed by SDS-PAGE or injected into animals for antibody production.


GuMCAC buffers (store 6 months at 4C)

GuMCAC-0 buffer:

20 mM TrisCl, pH 7.9
0.5 M NaCl
10% glycerol
6 M guanidineHCl

GuMCAC-500 buffer:

20 mM TrisCl, pH 7.9
0.5 M NaCl
10% glycerol
6 M guanidineHCl
0.5 M imidazole

GuMCAC-20, GuMCAC-40, GuMCAC-60, and GuMCAC-100 buffers:

These buffers (containing different concentrations of imidazole) are made by mixing GuMCAC-0 buffer and GuMCAC-500 buffer in the appropriate ratios: e.g., for GuMCAC-20 buffer, use 96:4 (v/v) GuMCAC-0/GuMCAC-500.

GuMCAC-EDTA buffer

20 mM TrisCl, pH 7.9
0.5 M NaCl
10% glycerol
6 M guanidineHCl
0.1 M EDTA, pH 8.0
Store 6 months at 4C

enjoy

[admin]

Excellent protocol Serg!

thanks

[microbalu]

hi there
r u following the instructions given by company (matrix) one has to optimize these processes like increasing the washes and keeping 20mM imidazole and 0.5 Mnacl protein:matrix ratio elution conditions ans so on.