Invitrogen™ ProBond™ Purification System

Catalog No.	Quantity
K85001	6 purifications

Catalog No. K85001 Supplier Invitrogen™ Supplier No. K85001

Description

Includes

6 x 2mL resin columns and buffers for native and denaturing purification; 12mL ProBond pre-charged resin

Designed for the purification of recombinant proteins that contain a polyhistidine (6xHis) sequence

• Utilizes proprietary ProBond nickel-chelating resin and is supplied with native and denaturing buffers for efficient purification of recombinant proteins under different conditions

His-Tagged Protein Purification, Protein and Peptide Purification, Protein Sample Preparation and Protein Purification, Proteins, Expression, Isolation and Analysis

Specifications

• Format: Suspension
• Product Type: ProBond™ Purification System
• Content And Storage: Six 2-ml resin columns and buffers for native and denaturing purification. Twelve milliliters of ProBond™ pre-charged resin. Store at +4°C. All reagents are guaranteed stable for 6 months when properly stored.
• Protein Tag: His Tag
• Quantity: 6 purifications
• Product Line: ProBond

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.

After I elute my protein from the ProBond column under denaturing conditions and I dialyze it to remove the urea, my protein precipitates. Any suggestions on how to correct this?

There are a few suggestions provided from our R&D scientists:

(1) Use stepwise dialysis against successively lower concentrations of urea buffers to slow the refolding.

(2) Add glycerol to the folding buffer; usually between 10 to 50%, but the amount must be determined empirically.

(3) After the denaturing washes, do several washes under native conditions and then elute under native conditions.

Note: you may also try to rescue a precipitated protein by adding denaturing buffer and either trying the glycerol dialysis or rebinding to the column.

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 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.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

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