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查看更多产品信息 ProBond™ Nickel-Chelating Resin - FAQs (R80101, R80115)
16 个常见问题解答
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis 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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
The enzyme could be denatured. Try buffer exchange or dialysis before digestion with EKMax Enterokinase.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
ProBond and Ni-NTA beads can be used in FPLC columns. However, the beads can only withstand low pressure (~43.5 psi max).
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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.