Invitrogen™ ProBond™ Nickel-Chelating Resin

Catalog No.	Quantity
50-255-9769	150 mL
R80101	50 mL

Catalog No. 50-255-9769 Supplier Invitrogen™ Supplier No. R80115

Description

A nickel-charged affinity resin used to purify recombinant proteins containing a polyhistidine (6xHis) sequence.

• ProBond™ Nickel-Chelating Resin is a nickel-charged affinity resin used to purify recombinant proteins containing a polyhistidine (6xHis) sequence
• Proteins bound to the resin may be eluted with either low pH buffer or by competition with imidazole or histidine
• One-step purification can be performed under both native and denaturing conditions
• ProBond™ Resin uses the chelating ligand iminodiacetic acid (IDA) coupled to a highly cross-linked 6% agarose resin
• Suitable for use in FPLC, batch, and gravity flow applications
• Guaranteed stable for 6 months when properly stored at 4°C

Specifications

• Content And Storage: The ProBond™ resin is pre-charged and able to bind 1-5 mg of recombinant protein per 1 ml of resin. It is provided as a 50% slurry in 20% ethanol. The resin will appear blue in color when charged with Ni²⁺ . Store at +4°C. ProBond™ resin is guaranteed stable for 6 months when properly stored.
• Stationary Phase: Nickel-Chelating
• Product Line: ProBond
• Type: Resin
• Quantity: 150 mL
• Form: Suspension
• Column Type: Affinity Column

Frequently Asked Questions (FAQs)

How do you typically detect expression of a recombinant fusion protein?

Western blot analysis is typically used to detect the expressed protein. We sell several antibodies against various epitopes, such as Xpress, HisG, V5, or C-terminal 6xHis. Additionally, His-tagged proteins can be purified using our ProBond Purification System via affinity purification.

What expression levels can be expected with the pTrcHis/CAT construct?

In one experiment, 35 µg CAT/mg total protein or 68.2 mg CAT/liter of culture was obtained. 50 ml of cell extract was loaded onto a ProBond column and 2 mg of CAT was recovered. The eluted protein appeared reasonably pure on a gel.

I purified my protein from a ProBond column using denaturing conditions. After elution, I tried digesting off my N- terminal tag with EKMax Enterokinase, but see no EK cleavage. What can you suggest I try?

The enzyme could be denatured. Try buffer exchange or dialysis before digestion with EKMax Enterokinase.

Can ProBond or Ni-NTA beads be used for large-scale preparations?

ProBond and Ni-NTA beads can be used in FPLC columns. However, the beads can only withstand low pressure (~43.5 psi max).

The pH of my ProBond buffers is off. Instead of pH 4-7, it is close to pH 9. What should I do?

pH drift is typical with these buffers. Adjust with concentrated HCl if the pH is too high or with 10 N NaOH if the pH is low.

My nickel resin turned white. Why is this?

A strong chelating agent like EDTA or EGTA has stripped the column and the Ni2+ has leached out. Use no more than 1 mM EDTA. We do not recommend reusing stripped resin.

My nickel resin turned brown. Why is this?

The nickel in the column is reduced. Avoid using DTT, buffers with amines in solutions (Tris, HEPES, MOPS) and certain amino acids (arginine, glutamine, glycine). If the sample needs to be reduced, 20 mM beta-mercaptoethanol can be used.

In order to recharge the column, the Ni2+ must first be stripped away. We recommend incubating the column in 1M HCl for 1 hour at room temperature. At this point, the column should turn from brown to colorless. Wash with water to remove acid and add 50 mM NiSO4 to recharge the column. At this time, the column should return to its normal blue color.

My His-tagged protein is degraded after isolation using the ProBond Purification System. Do you have any suggestions on how to alleviate this?

The His-tagged protein may be an N-terminal tagged protein. Protein degradation during expression may not allow the expression of the full-length. If this is the case, an alternative is C-terminal His-tagged expression. If additionally, the expression is done in T7-promoter driven vector such as pET vector, induce with only 0.5 to 0.7 mM IPTG instead of 1 mM. Lastly, try to work at 4 degrees C at all times and use protease inhibitors during lysis.

Do you have a protocol for purification of His-tagged proteins from Pichia lysates?

Alcohol oxidase (AOX protein) is an octamer and has at least a few His stretches. Hence, AOX protein will bind to the ProBond resin. In order to prevent co-elution, we recommend that you perform ion exchange purification prior to the ProBond purification. You will need to know the pI of the expressed protein for good binding and need to optimize the ion exchange step for efficient separation from AOX.

We recommend purifying His-tagged Pichia proteins using the protocol described on Pages 50 and 51 in the Pichia manual (https://tools.thermofisher.com/content/sfs/manuals/easyselect_man.pdf), under Purification. It describes how to obtain the supernatant (soluble proteins) and pellet (urea/insoluble proteins) by using the breaking buffer (BB). The composition of the breaking buffer is listed on Page 59 of the Pichia manual.

Do you have any suggestions for purification of His-tagged proteins from S. cerevisiae?

Over-expressed S. cerevisiae proteins should be purified as described on Pages 50 and 51 in the Pichia manual, (https://tools.thermofisher.com/content/sfs/manuals/easyselect_man.pdf; see the section under Purification). However, if the breaking buffer is used for S. cerevisiae, the EDTA should be omitted. In other words, if working with Pichia, the breaking buffer should contain ETDA (see Page 59 of the Pichia manual for the Breaking Buffer recipe), but EDTA should be omitted if working with S. cerevisiae.

Do you have suggestions for purification of secreted His-tagged proteins from E. coli culture?

A simple and reliable method for precipitating protein from bacterial culture supernatants based on a pyrogallol red- molybdate-methanol (PRMM) protocol has been developed and applied for the analysis of proteins secreted by a bacterial type III secretion system (Caldwell RB, Lattemann CT (2004). Simple and reliable method to precipitate proteins from bacterial culture supernatant Appl Env Micro 70(1):610-612)(http://aem.asm.org/content/70/1/610.full.pdf).

Do you have a protocol for purification of His-tagged proteins from E. coli lysate?

When purifying His-tagged proteins proteins from E. coli lysates, keep in mind that there is a 29 kDa endogenous protein SlyD. SlyD has a histidine-rich C-terminus and is found in all strains of E. coli and Salmonella. The contamination is apparent when the His-tagged protein is expressed at a low level or not expressed at all. In such cases, SlyD will bind to the nickel column with great affinity. Increase the purification stringency to overcome SlyD binding.
If protein is released into LB media from E. coli, try native isolation conditions. Dialyze against binding buffer and possibly concentrate before going on to the ProBond resin (10% glycerol in the dialysis binding buffer will concentrate the secreted protein well). Another option is to add about 24 g NaCl and 2.8 g Na2HPO4 per liter of media, and adjust the pH to 7.8 with NaOH or HCl. This will turn the media into pseudo-binding buffer (~500 mM NaCl, ~20 mM NaPO4, pH 7.8); perform binding, washing, and eluting with either imidazole or by altering pH.

Is the ProBond resin compatible with FPLC?

Yes, the maximum flow rate is 4 mL/hr (1 mL column) and the maximum linear flow rate is 700 cm/hr in an XK16/60 column with a 5 cm bed height. We have successfully used a flow rate of 2 mL/min. The maximum pressure is 0.3 MPa (3 bar, 42 psi). Do not run above 50-100 psi. The pH stability for long term is 3-13 and 2-14 for short term.

What are the specifications for the ProBond purification columns you offer?

The columns are 9 cm high, conical 0.8 x 4 cm of polypropylene and hold up to 2 mL of resin and 10 mL of eluent or sample. The average pore size of the column filter is 30-35 microns.

What should the typical protein recovery be when using the Probond Purification System or Ni-NTA Purification system?

Both systems are qualified by purifying 2 mg of myoglobin protein on a column and performing a Bradford assay. Protein recovery must be 75% or higher.

For Research Use Only. Not for use in diagnostic procedures.