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View additional product information for ProBond™ Nickel-Chelating Resin - FAQs (R80101, R80115)
33 product FAQs found
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.
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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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Consider an additional high stringency wash at a lower pH, i.e., between pH 6-4 prior to the elution. This applies for denaturing or hybrid purification. For native purification, the pH should only be decreased a little (exact pH depends on the pH optimum of the protein) in order to maintain protein structure.
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Try stringent purification methods including:
-Increase NaCl concentration to 2 M or greater. This should be tried first because the salt can be easily removed via dialysis.
-Increase the imidazole concentration
-Decrease the pH; for native purification, this can be reduced as long as protein function can be maintained. If not, decreasing pH should be tried anyway under such circumstances because protein can be refolded later if the native purification method does not achieve elution.
If the protein does not elute despite stringent elution procedure, the column must be stripped by one of the following methods:
-DTT: 1 mM or greater should be used.
-EDTA or EGTA: incubation of resin in 10-100 mM should be sufficient.
Note: Proteins should come off as long as they don't precipitate on the column. Stripping the column will cause resin contamination of the protein. Either dialysis or centrifugation can remove the stripped resin. The stripped resin should not be reused again.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
-Increase stringency with NaCl and imidazole. Do an imidazole step gradient to elute the His-tagged protein.
-Decrease the pH by 0.5 -1.0 pH unit during denaturing purification. This can also be tried during native purification.
-Add beta-ME (beta-mercaptoethanol) to a maximum of 20 mM to reduce the disulfide bond.
-If too much resin is being used, the binding capacity exceeds the amount of His-tagged proteins. In this situation, endogenous proteins containing metal binding sites will bind to the resin with increased affinity.
-Co-elution of non-tagged proteins can be eliminated by doing a second round of purification from the eluate. Thus, dialyze against the binding buffer with 10-20 mM imidazole. This will significantly reduce the nonspecific binding capacity.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
Please see our suggestions below:
-Add 0.1% Triton X-100 or Tween-20 to help solubilize further; purify at room temperature if protein is not temperature sensitive. However, keep in mind that most proteins are temperature sensitive.
-If secreted proteins are in media with low pH, they must be dialyzed to avoid Ni2+ reduction.
-If solubility is a real problem (e.g., microsomes), include up to 0.2% Sarkosyl in the 6 M guanidine lysis buffer; this will help to solubilize everything and may be still compatible with the ProBond or Ni-NTA purification column.
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The protein may have been eluted because the procedure was too stringent. Reduce the stringency as follows:
-Increase the pH by 0.5 to 1.0 pH unit
-Decrease the NaCl concentration in increments of 50 to 100 mM
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The lack of binding could be because the protein already eluted with the wash buffer. This means that the purification conditions were too stringent. To achieve less stringent conditions, try these options:
-Use only 10 mM or less imidazole (1-5 mM) in the binding or wash buffer and /or
-Reduce the NaCl concentration from 500 mM to 250 mM or less; a systematic titration may be necessary, i.e., reduce in increments of 100 mM and then fine tune.
-Try to use an imidazole step gradient.
-The His-tag is hidden due to folding; try a denaturing elution.
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Chromosomal DNA may be the cause. Ensure that genomic DNA is sheared by pulling the lysate up and down a few times through an 18-gauge needle. Ensure further that the lysate is spun for 15 min at 10,000 rpm in a Sorvall SS-34 rotor in order to clear the lysate. Filtration of the lysate through a 0.8 µm filter is another option.
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Try heating the buffer to 37 degrees C. The precipitate should re-dissolve back into solution. If heating does not help, check the pH. It should be between pH 7.8-8.0, and can be adjusted with HCl or NaOH.
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Unfortunately, if the resin completely froze, then it might not efficiently bind to proteins anymore. A good way to check the quality would be to let the resin thaw. If the resin forms aggregates or clumps, the resin is no longer functional. If you do not see clumps or aggregates, then try testing the resin on a non-precious sample.
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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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This can be achieved, though not effectively. DMEM and other mammalian tissue culture media contain many contaminating proteins (and free amino acids like glutamine and histidine) that may compete for binding to the ProBond resin. For best results, mammalian tissue culture media should be dialyzed (against binding buffer) or filtered prior to loading on equilibrated ProBond resin. Other methods such as ion exchange or sizing columns can help to prepare the protein for effective isolation on the ProBond column. If necessary, the culture media may be adjusted to facilitate direct binding to the column. The pH of the media should be adjusted to 7.5-8.0. The salt concentration in the media should be 0.5 M NaCl.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
Up to 6 M guanidinium chloride or up to 8 M urea can be used.
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Please see the suggested reagents below:
-Up to 60 mM citrate has been used successfully
-Up to 20% ethanol prevents hydrophobic interaction between proteins
-Up to 50% glycerol prevents hydrophobic interaction between proteins
-Imidazole can be used at low concentrations (20 mM) to inhibit nonspecific binding and at higher concentrations (>100 mM) to elute the 6xHis-tagged proteins.
-Up to 2 M sodium chloride prevents ionic interactions; you should use at least 150 mM
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Non-ionic detergents including Triton, Tween, NP-40 detergents can be used at up to 2% concentration to remove background proteins and nucleic acids. Cationic detergents can be used at up to 1% concentration. Anionic detergents such as SDS or Sarkosyl detergent are not recommended. Zwitterions (such as CHAPS) can be used at up to 1% concentration.
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EDTA and EGTA can be used at up to 1 mM concentration. Higher concentrations will strip the resin (i.e., the nickel ion will leach out).
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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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Please keep in mind, while the ProBond resin is rechargeable, the Sepharose medium will sustain wear. If the resin turns white due to the loss of nickel ions from the column, recharge the resin.
To recharge 2 mL of resin in a purification column:
1.Wash the column two times with 8 mL 50 mM EDTA to strip away the chelated nickel ions.
2. Wash the column two times with 8 mL 0.5 M NaOH.
3. Wash the column two times with 8 mL of sterile, distilled water.
4. Recharge the column with two washes of 8 mL NiCl2 hexahydrate at a concentration of 5 mg/mL prepared in sterile, distilled water.
5. Wash the column two times with 8 mL distilled water.
6. Add 0.02% azide or 20% ethanol as a preservative and cap or apply a Parafilm cover to the column. Store at room temperature.
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ProBond resin can be used for up to three or four purifications of the same protein without recharging. We only recommend reusing the resin for purification of the same recombinant protein. If you want to reuse the resin, wash it with 0.5 M NaOH for 30 minutes and equilibrate the resin with the appropriate binding buffer.
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We have purified proteins with a minimum of 6 histidines. Some literature has indicated purification from as little as 4 or 5 histidines.
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The ProBond resin is coupled with iminodiacetic acid groups (IDA). IDA is loaded with Ni2+ ions and binds Ni2+ by three coordination sites. The resin is used to purify 6x His-tagged overexpressed proteins. One mL should bind at least 1 mg of recombinant protein.
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ProBond resin (Cat. No. R80101) is available separately but the empty plasmid columns (Cat. No. 1954124, 1954126) have been discontinued.
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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.
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