ProBond™ Purification System - FAQs

通常使用免疫印迹分析法检测蛋白表达。我们提供许多针对不同表位的抗体，如Xpress、HisG、V5或C-端6xHis。此外，可以用我们的ProBond亲和纯化系统来纯化His标签蛋白。

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.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

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.

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

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

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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.

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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.

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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.

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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.

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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).

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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.

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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.

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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.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.