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Probond and Ni-NTA resin
TABLE OF CONTENTS
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Product Specific Information
Probond purification
Denaturing purification
Native purification
Hybrid purification
Refolding protein
Determining protein concentration
Recharging resin
Column and resin properties
Columns
Resin
Ni-NTA vs IDA
Other heavy metals for charging columns
Buffer and Reagent compatibilities
Buffers
Chelating reagents
Sulfydril reagents
Detergents
Amino acids and native elution agents
Other reagents
Other additives
Denaturants
Troubleshooting if purification does not work well
Resin does not settle down
Native eluted fractions do not contain tagged protein
Denatured eluted fraction does not contain tagged protein
Protein precipitation during native binding
Eluted protein contaminated with endogenous proteins
Protein does not elute
Protein expression is low
His-tagged protein is degraded
Inclusion bodies
Protocol specific information
Protocol for purification of His-tagged proteins synthesized using Expressway
Protocol for protein-protein interaction studies
Protocol for purification from E. Coli lysate
Protocol for purification from S. Cerevisiae
Protocol for purification from Pichia lysates
Protocol for purification from baculovirus lysates
1
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
RELATED TECHNICAL SUPPORT NOTES
2
PRODUCT DESCRIPTION
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The Probed 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.
Ni-NTA Agarose uses nitrilotriacetic acid (NTA), a tetradentate chelating ligand, in a highly cross-linked 6%
agarose matrix. NTA binds Ni2+ ions by four coordination sites. This resin is also used to purify 6x His-tagged
overexpressed proteins, see further details in under 鈥淧robond Resin鈥?.
Proteins can be expressed in E. coli, Yeast, insect or mammalian cells. The proteins can be purified under
denaturing, native or hybrid conditions. Hybrid conditions means to start purification under denaturing conditions
and then changing to native conditions.
SHIPPING CONDITIONS
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The purification systems are sent out on wet ice.
STORAGE CONDITIONS
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Buffers and solutions are stored at room temperature or 4掳C鈥攖he manual states room temperature but the labels
state 4掳C. Both temperatures are fine (except there may be some precipitate in buffers at 4掳C, which can be
resolved by heating the buffer to 37掳C).
Do not freeze resin. Unfortunately, if the resin completely froze then it might not efficiently bind to proteins
anymore.
If freezing is suspected, 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. This is what occurs when the resin is frozen at -20oC.
However, if you observe no clumping/aggregates then recommend testing the resin on a non-precious sample.
All reagents are guaranteed stable for 6 months when properly stored.
QC SPECIFICATIONS
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The ProBond Purification System and Ni-NTA are qualified by purifying 2 mg of myoglobin protein on a
column and performing a Bradford assay. Protein recovery must be 75% or higher.
PROTOCOL AND APPLICATION NOTES
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Product specific information
Probond purification
Denaturing purification
Native purification
Hybrid purification
Refolding protein
Determining protein concentration
Recharging resin
Column and resin properties
Columns
Resin
Ni-NTA vs IDA
Other heavy metals for charging columns
Buffer and Reagent compatibilities
Buffers
Chelating reagents
EDTA, EGTA
3
Sulfydril reagents
Detergents
Amino acids and native elution agents
Other reagents
Other additives
Denaturants
Troubleshooting if purification does not work well
Protocol specific information
Protocol for purification of His-tagged proteins synthesized using Expressway
Protocol for protein-protein interaction studies
Protocol for purification from E. Coli lysate
Protocol for purification from S. Cerevisiae
Protocol for purification from Pichia lysates
Protocol for purification from baculovirus lysates
Probond purification requires five steps that are outlined briefly below
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Wash the resin with water and then 4 column volumes of binding buffer
Lyse the cells
Harvested cells are resuspended in the Lysis buffer and lysed. Lyse e.g through sonification or homogenization.
The lysate is then passed through an 18 gauge needle in order to sheare DNA. A 3000x g centrifugation step
follows and a cleared supernatant or lysate is yielded.
Bind The cleared cell lysate is added to the resin; resin and lysate are mixed gently at +4oC for ~30 min.
Wash the column with the wash buffer to remove all non-specific-binding proteins. Wash with 4-5 column
volumes.
Elute the His-tagged protein with Imidazole or the acidic pH: 4.0. For instance collect 1 ml fractions.
Remember that all buffers are sodium phosphate buffers and 500 mM NaCl.
Note: Some literature may indicate purification from 4 or 5 histidines. We have not tried to purify proteins with
less than six histidines
Denaturing Purification (pH: 7.8 to 6.0 to 4.0)
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Gu-HCl and Urea are strong denaturants that solubilze inclusion bodies. Insoluble proteins are trapped in inclusion
bodies, which are associations of unfolded proteins). Insoluble proteins tend to precipitate during purification are
purified
Lysis buffer (pH: 7.8) contains 6 M Guanidine HCl (Gu-HCl) and 0.5 M NaCl.
Binding buffer (pH: 7.8) contains 8 M Urea and 0.5 M NaCl.
Wash buffer (pH: 6.0) contains 8 M Urea and 0.5 M NaCl
Elution buffer (pH: 4.0) contains 8 M Urea and 0.5 M NaCl; Histidine becomes protonated at low pH (~ 4-5);
thus, it can no longer bind to the resin; 鈥淚t falls off the column鈥?. One can add 10 or 20 mM Imidazole to Lysis-,
Binding,- and Wash buffer to increase stringency.
Native Purification at ~ pH: 8.0 鈥? Imidazole concentration
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Lysis buffer and the Binding buffer are the same! They contain 0.5 M NaCl and 10 mM Imidazole.
Wash buffer contains 0.5 M NaCl and 20 mM Imidazole
Elution buffer contains 0.5 M NaCl and 250 mM Imidazole
All buffers are made from the 5x native purification buffer by mixing 80 ml water with 20 ml 5x buffer to yield
1x native purification buffer. The pH should be around 8.0 and can be either adjusted with HCl or NaOH
4
Various Imidazole concentrations are added to yield the respective buffer.
Imidazole elution at pH 8.0 can also be done in a stepwise manner. Start out for instance with 10 mM then 50mM,
100 mM, 150, 200, 250, 300 mM until the 鈥減rotein falls off鈥?. Keep in mind that more contaminating proteins that
would have been eliminated at pH 6.0 will remain on the resin at pH 7.0. If the protein function is not impaired at
pH 6.0, one can wash and elute with Imidazole at pH 6.0.
Hybrid Purification
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Lysis and binding are done with the respective denaturing buffers. The washing and elution step, however, are
continued with the respective native buffer (One also can do the switch during the washing; do denaturing washing
steps, switch to one or two native washing steps and elute with the native elution buffer).
Reason for Hybrid Purification: The protein is insoluble, but the refolding is necessary to yield a functional protein
for the activity assay. Instead of the hybrid method, one can purify entirely denaturing and refold later, see below.
This is recommended if large yields are required.
Refolding protein following purification
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Maintain low protein concentrations 10-50 ug/ml.
Disulfide bonds help to stabilize native proteins; add a redox pair such as GSH (reduced glutathione) and
GSSG (oxidized Glutathione at a ratio of 10:1 with GSH concentration of 2-5 mM. Such a redox pair is helpful
to create an oxidizing potential to break and build new S-S bonds during the folding Process.
Remove denaturants slowly by dilution or dialysis; Glycine (50 mM, pH 9.0, 5 mM EDTA) helps to
solubilize the proteins. If GuHCl (Guanidinium HCl) is used, add 2 M urea because Urea helps to stabilize the
protein folding, too.
Add detergents at a very low concentration such as 0.1-0.5% NP40 or 0.005% (v/v) Tween 20.
Include co-solvents to stabilize the proteins such as glycerol (5-20%) or PEG 8000 or glucose or sucrose (10
%)
Certain anions (e.g. phosphate or sulfate) or cations (e.g. MES or HEPES) have positive effects, too.Include
salt and maintain a neutral pH such as 100 mM KCl, or 150-500 mM NaCl, 2 mM MgCl2
Avoid protein degradation by adding protease inhibitors such as 0.5 mM PMSF, 0.005-2 ug/ml aprotinin, 2
ug/ml
Pepstatin, or 2-5 ug/ml leupeptin.Add detergents at a very low concentration such as 0.1-0.5% NP40 or 0.005%
(v/v) Tween 20.
Include co-solvents to stablize the proteins such as glycerol (5-20%) or PEG 8000 or glucose or sucrose (10%).
Certain anions, e.g. phosphate or sulphate or cations such as MES or HEPES have positive effects.
Dialysis of a phosphate buffer when against calcium will result in a calcium phosophate precipitate. If CaCl2 is
needed for subsequent enterokinase digestion (10mM Tris pH 8, 10mM CaCl2), remember to add CaCl2 after
dialysis is complete.
Determining protein concentration in buffers containing imidazole, pH: 7.0
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Quant-iT protein assay kit from Molecular Probes with cat# Q33210 1.25 mM
This fluorescence-based protein assay is easier and more sensitive than standard absorbance-based assays (for
sample concentrations from 12.5 碌g/mL to 5 mg/mL) Quantitates proteins from 0.25 碌g to 5 碌g
Signal is unaffected by many common contaminants , including DTT, beta-mercaptoethanol, amino acids, and DNA
Little protein-to-protein difference in signal. Signal is stable for 3 hours
Imidazole at a final concentration below 1.25 mM is acceptable. Above that concentration, the imidazole begins to
interfere. As always, the most accurate results will be achieved by adding the same concentration of imidazole to the
standards as well (Molecular Probes Technical Support, Wayne Considine, June 05).
5
Imidazole does absorb at 280nm, and the absorbance varies with concentration. So to be perfectly accurate each
eluted fraction should be blanked against its elution buffer. To give an idea of the level of absorbance various
imidizole concentrations in 20mM NaPhosphate, 500mM NaCl, pH 6.0 where checked.
Imidizole Concentration OD280
50mM 0.030
100mM 0.064
200mM 0.132
350mM 0.237
500mM 0.338
Recharging Resin
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Theoretically, the resin is infinitely rechargeable, but the sepharose will undergo wear out.
The resin is good for three or four purifications of the same protein without recharging.
We have recharged ProBond resin here more than three times and still had good binding and elution.
We use NiCl2 鈥? 6H2O Hexahydrate 5 mg/ml. We have not tried Ni SO4, but it is referenced in the literature
often.
COLUMN AND RESIN PROPERTIES
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Columns
Resin
Ni-NTA vs IDA
Other heavy metals for charging columns
Columns
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Columns are 9 cm high, conical 0.8 x 4 cm made of polypropylene and hold up to 2 ml of resin and 10 ml of eluent
or sample.
Average pore size of column filter is 30-35 microns
Probond Resin
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The Probond resin is a sepharose resin that is coupled with iminodiacetic acid groups (IDA). IDA is loaded
with Ni2+ ions and binds Ni2+ by three coordination sites
Average size of resin particle is 90 microns. The particle size range is 45-165 microns
The resin has a net positive charge therefore negatively charged proteins regardless of whether they have the
His6 tag- will bind to the resin nonspecifically. 500mM NaCl negates this effect. The salt concentration should
always be 0.5 - 1M to avoid ion exchange effects..
Recommended flow rate: 0.5 ml/min
Kd of His for IDA: 10-11 to 10-13 Kd range.
Immobilized copper or nickel ions bind native proteins with a Kd of 1-17 x 10-5 M (Hutchen and Yip,
1990). Addition of a histidine tail results in a protein that binds to the Ni2+-NTA complex with a Kd of 10-
13
M at pH 8.0 even in the presence of detergent, ethanol, 2 M KCl, 6 M guanidine, or 8 M Urea. (I could
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not find a Kd specifically for IDA, but it should be an ESTIMATE.)
(This info was taken from Current Protocols in Mol Bio, Chapt 10.11.21)
Binding capacity of probond resin: One ml should bind at least 1 mg of recombinant protein, could be more
depending on the protein = 1 to 5 mg of recombinant protein per 1 mL of bed volume.
Temperature of binding can be 4 deg C or RT. higher temps have not been tried and may result in protein
degradation.
Some literature may indicate purification from 4 or 5 histidines. We have not tried to purify proteins with
less than six histidines.
Probond resin is FPLC compatible, and so is the Qiagen Ni-NTA, see below.
The maximum flow rate is 4ml/hr (1ml column) and the maximum linear flow rate is 700 cm/hr in an XK16/60
col with a 5cm bed height. We have successfully used a flow rate of 2 ml/min.
The max 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.
One customer ran 1 to 2 liters over a 20 ml ProBond column at 0.4 ml/min (It will take many hours).
Difference between IDA and NTA
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NTA binds Ni2+ ion by four coordination sites.
IDA binds Ni2+ by 3 coordination sites.
Because nickel is bound by 4 instead of 3 of its coordination sites, NTA resin minimizes leaching of the metal
from the solid support and allows for more stringent purification conditions.
Other heavy metals for recharging resin
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The most common metals used for Immobilized Metal Chelating Chromatography (IMAC) include Cu(II), Ni(II),
Ca(II), Zn(II), Fe(II), and Fe(III). However, other group III metals have been studied. The sorbent is prepared by first
coupling a chelator, such as iminodiacetic acid (IDA), via a spacer arm to the support, followed by chelation with the
metal of choice. This interaction is specific and strong due to the nitrogen and two oxygen ligands of the IDA that form
the metal coordination complex.
To immobilize the metal, 2 mL of 0.2 M metal ion solution in water is injected onto the column immediately after an
EDTA/distilled water wash. Excess metal can be washed out with 5 mL of deionized water. The column must next be
equilibrated with 15 mL of the weak buffer followed by a blank gradient before chromatographic analysis is attempted.
After the metal is coordinated with the IDA, it has three sites available for specific protein surface electron donor atoms.
The chelated metal may be reomved from the column using EDTA and replaced with another metal after the column has
been washed to remove any remaining EDTA.
Each metal has its own specificity with respect to protein retentation. The choice of metal is protein specific. An
examination of standard protein mixtures on IMAC revealed that the affinity and resolution of the test protein separation
s for immobilized metal ions followed the order Cu2+ > Ni2+ > Zn2+. It is thus recommended, unless existing information
regarding an alternate metal and the target protein is available, that initial separations be attempted using an immobilized
Cu(II) column.
(From: Methods in Enzymology, Vol 182, "Guide to Protein Purification", Edited by Murray P. Deutscher, pgs 419-420)
Buffer & Reagent compatibilities of Ni-NTA and the ProBond
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Buffers
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Chelating reagents
Sulfydril reagents
Detergents
Amino acids and native elution agents
Other reagents
Other additives
Denaturants
Buffers
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Tris HCl, HEPES, MOPS PIPES Up to 100 mM have been used in some cases, but one should only
recommend 50mM Buffers. REASON:
Others used successfully: try to find concentrations ~ 100 mM should be fine:
Tris-Phosphate
Tris-Acetate
Sodium Acetate
Sodium Borate
Mes-NaOH
Pipes-HCl
Chelating reagents
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EDTA, EGTAUp to 1 mM have been used in some cases. Higher concentrations will strip the resin, i.e. the Ni ion
will leach out.
Sulfhydril reagents
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beta-mercaptoethanol; Use up to 20 mM can be used.
DTT, DTE Low concentrations will reduce nickel ions. No more than 1 mM DTT or DTE should be used.
Otherwise, the column will be stripped.
Detergents
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Nonionic detergents (Triton, Tween, NP-40, etc.) Removes background proteins and nucleic acids. Up to 2%
can be used.
Cationic Up to 1% can be used (will tie up nickel ions and prevent binding)
Anionic SDS, Sarkosyl. Not recommended, but up to 0.2% has been used successfully in some cases
Pluronic F68 surfactant found in insect media will strip the column in some cases. Test first. If stripping,
Media should be dialyzed first.
Zwitterions (such as chaps) Up to 1% CHAPS has been used successfully in some cases
Amino acids / Native Elution Agents
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Glycine is not recommended because it out-competes His
Glutamine is not recommended because it out-competes His.
Arginine should not be recommended because it out-competes His. (0.2 mM Arginine have been used
successfully in some cases)
Histidine Can be used at low concentrations (20mM) to inhibit non-specific binding and at higher
concentrations (>100 mM) to elute the 6xHis-tagged proteins.
Other reagents to prevent non-specific binding
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Citrate Up to 60 mM has been used successfully
Ethanol Prevents hydrophobic interaction between proteins Up to 20%
Glycerol Prevents hydrophobic interaction between proteins Up to 50%
Imidazole Can be used at low concentrations (20mM) to inhibit non-specific binding and at higher
concentrations (>100 mM) to elute the6xHis-tagged proteins.
Sodium Chloride Prevents ionic interactions Up to 2 M, should use at least 150 mM
Other Additives
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Ammonium: Not recommended; 10 mM Ammonium Sulfate has been used successfully in some cases
Calcium: 5 mM has been successfully in some cases; it will displace the Nickel ion from the column
Sodium bicarbonate Not recommended
Hemoglobin: Not recommended
Magnesium chloride will not interfere since it doesn鈥檛 bind ProBonds IDA. Up to 4M tested
Sodium azide as antimicrobial will work, but 20% EtOH is standard for antimicrobial.
Denaturants
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Gu HCL Up to 6 M
Urea Up to 8 M (Probond works better than NiNTA on denatured fractions from E Coli lysate but NiNTA
seems to work better on the soluble fractions.
TROUBLESHOOTING鈥? IF PURIFICATION DOES NOT WORK WELL
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Resin does not settle down
Native eluted fractions do not contain tagged protein
Denatured eluted fraction does not contain tagged protein
Protein precipitation during native binding
Eluted protein contaminated with endogenous proteins
Protein does not elute
Protein expression is low
His-tagged protein is degraded
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Inclusion bodies
How to avoid inclusion bodies
Purification of protein from inclusion bodies (Method A and Method B)
Resin does not settle down
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Chromosomal DNA may be the cause. Ensure that genomic DNA is sheared by pulling up and down the lysate a few times
through a 18-gauge needle. Ensure further the lysate is spun for 15 min at 10000 rpm in a Sorval SS-34 rotor in order to clear
the lysate. Filtration of the lysate through a 0.8 um filter is another option.
Native eluted fractions do not contain the His-tagged protein
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They did not bind because the protein eluted already with the wash buffer. It means the purification conditions were too
stringent. In order to be less stringent, try to do the following:
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 an Imidazole gradient step gradient.
The His-tag is hidden due to folding; try denaturing elution.
Denatured eluted fraction does not contain the His-tagged protein
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The protein has been washed out 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] in increments of 50 to 100 mM.
Protein precipitation during native binding
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Add 0.1% Triton X-100 or Tween-20 to help to solubilized further; (eventually purify at room temperature if
protein is not temperature sensitive. However, most proteins are temperature sensitive).
If secreted proteins are in media with low pH, they must be dialysed to avoid Ni2+ reduction.
If solubility is a real problem (e.g. microsomes) include up to 0.2% Sarkosyl in the 6M Guanidine lysis buffer鈥?
The will help to solubilize everything and may be still compatible with the Probond or Ni-NTA purification
column.
Eluted protein is contaminated with endogenous proteins
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Increase stringency with NaCl and Imidazole. Do an Imidazole step gradient to elute the His-tagged protein.
Decrease the pH if 0.5 -1.0 pH unit during denaturing purification. This can also be tried during native
purification.
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Add beta-ME (beta-mercaptoethanol) to the maximum of 20 mM to reduce the S-S bond.
If too much resin is being used, the binding capacity exceeds the amount of His-tagged proteins. Hence,
endogenous proteins with metal binding sites bind with increased affinity.
Co-elution of non-tagged proteins can be eliminated by doing a second round of purification from the eluate.
Thus, dialyse against the binding buffer with 10-20 mM Imidazole. This will reduce significantly reduce the
non-specific binding capacity.
Protein does not elute
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If the protein does not elute despite stringent elution procdure, the column must be stripped by one of the following methods:
Proteins should come off as long as they don鈥檛 precipitate on the column.
DTT at 1 mM or greater should be used.
EDTA or EGTA Incubation of resin in 10-100mM should be sufficient.
REMEMBER: Before stripping the stripping the column, try stringent purification methods first such as:
Increase [NaCl] 2 M or greater. This should be tried first because the salt can be easily removed via
dialysis.
Strippng 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.
Increase the Imidazole concentration during native purification; can also be used for denatruing purification.
Decrease the pH during denaturing purificiation; can also be tried during native purification 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 allow any elution.
Protein expression is low
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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 denturing or hybrid purifiction. In case of native purification, the stringency can be increased as outlined
above. For native purification, the pH should only decreased a little (exact pH depends on the pH optimum of the protein) in
order to maintain protein structure.
His-tagged protein is degraded
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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, the customer should try the C-terminal His-tagged expression. If addtionally,
the expression is done in T7-promoter driven vector such as pET, the customer should induce with only 0.5 to 0.7 mM IPTG
instead of 1 mM.
Inclusion bodies
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Often, overexpression leads to the production of inclusion bodies that are insoluble aggregates of misfolded protein. These
inclusion bodies can easily be purified. However the solubilization of the expressed protein can usually only be obtained
using strongly denaturing conditions.
How to avoid inclusion bodies
11
Purification of protein from inclusion bodies (Method A and Method B)
How to avoid inclusion bodies
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Promoter Variation - Try different promoters to alter expression. Sometimes a slightly lower expression level
will prevent inclusion body production
Sorbitol/Betaine Method: Vary the amount of Sorbitol in growth media to alter osmotic pressure; reference
seems to be unknown. Grow in media containing 2.5 mM Betaine and try various amounts of Sorbitol (330
mM, 660 mM, 1 M for instance) and check for inclusion body production. Filter sterilize Betaine and Sorbitol
solutions and add to the autoclaved LB (reason: not sure if Sorbitol or Betaine can be autoclaved).
3.3 M Sorbitol
60.6 g in 100 mL water
0.5 M Betaine
7.08g in 100 mL
Cell Strain Variation Transform in many different cell strains with the offending plasmid.
DH5alpha F鈥? /endA1 hsdR17 (rK- mK+) glnV44 thi-1 recA1 gyrA (Nalr) relA1 delta (LacIZYA-argF)
U169 deoR (phi80dlac delta(lacZ)M15)
JM109
GM48 F- thr leu thi lacY galK galT ara fhuA tsx dam dcm glnV44
TOPP1-TOPP6
BL21 (DE3) F- ompT gal [dcm] [lon] hsdSB (rB- mB-; an E. coli B strain) with DE3, a lambda prophage
carrying the T7 RNA polymerase gene
BL21 pLys
W3110
NM522
N4830-1
Purification of Proteins from Inclusion bodies
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Method A Sarkosyl Method
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In this method cells are lysed through sonication in the presence of lysosyme. After centrifugation pelleted protein
(inclusion bodies) is solublized with 1 % sarkosyl.
Materials:
STE Buffer 100 mM NaCl
10 mM Tris-HCl pH 8.0
1 mM EDTA
STE-S STE Buffer (1 % Sarkosyl)
STE-T STE Buffer (1 % Triton X-100)
Procedure- This procedure is written for a 150 mL culture you may scale it up or down accordingly.
Cells from 150 mL culture were harvested in the Beckmann J2-21 in a JA-20 rotor at 5000 rpm for 15 minutes
@ 4掳C.
Resuspend the resulting pellet in 3 mL of STE buffer.
Add lysozyme to a final concentration of 150 碌g/mL and place on ice for 30 minutes.
Sonicate on ice, using a 40 second sonication and a 20 second rest period (set to 40 and use the small probe).
Repeat an additional three times.
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Aliquot into two 1.5 mL microfuge tubes.
Spin for 5 minutes @ 4掳C on maximum. (Keep the supernatant for further analysis).
Resuspend the pellet in 0.1 mL of STE-S. This will require vigorous pipetting (the pellet may not be completely
soluble).
Centrifuge for 2 minutes at 4掳C and transfer the supernatant to a 1.5 mL microfuge tube.
Add 9 volumes of STE-T to the supernatant (to form a mixed-micelle with sarkosyl).
Method B
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The essence of this method is to purify the inclusion bodies followed by selective solubilization of the expressed
protein.
Procedure- This protocol was designed for a 1L culture. However, one can scale the volumes up or down,
depending on the type of experimentation performed. Both 100ml cultures and 500 ml cultures were prepared for
this protocol (the results section specifies further).
The cells from the 1L culture are transformed into two 500ml tubes and are pelleted by centrifugation at
5000rpm/min at 4 degrees C.
To prevent contaminating the inclusion bodies by proteins from the outer membrane, the outer membrane is
removed by resuspending the cell pellet from 1L culture in 50ml BUFFER A. (If 100ml culture was prepared,
only 5 ml Buffer A would be required. This down -scaling of volumes is required whenever a volume is
indicated).
Incubate on ice for 10 minutes. Pellet the cells by centrifugation at 6000rpm/5min at 4 degrees C. Resuspend in
50ml of ice cold water. Leave on ice for 10 min. Centrifuge at 8200 rpm/5 min at 4 degrees C.
Resuspend the pellet in 10ml Buffer P containing protease inhibitors.
Sonicate the resuspended mixture 3x (50W) each with a 30sec. pulse with a 30 sec. pause in between pulses.
RNase T1 (1.3x103 U/10ml) + DNase I (400 ug/10ml) are added to the sonicated cell suspension and incubated
at room temp for 10 min.
The suspension is further diluted by addition of 40 ml of Washing Buffer P and the crude inclusion bodies are
pelleted by centrifugation at 11,000 rpm for 30 min at 4 degrees C. Keep aliquot and assay the supernatant for
proteins.
The pellet (inclusion bodies) is suspended in 40 ml of Buffer W. Incubate on ice for 10 min and centrifuge at
15,000 rpm/5min at 4 degrees C. for 10 min. Keep aliquot of supernatant. Repeat this washing step and save the
supernatant.
Resuspend the pellet in 10ml of Buffer D. A brief sonication with a 5 sec. pulse (50W) facilitates the
solubilization of the aggregated proteins (NOTE: the pellet may also be resuspended in 4M or 8M urea in
addition to BUFFER D).
The resultant suspension is incubated on ice for an additional 1 hour.
Centrifuge at 10,500 rpm for 30 min at 4 degrees C. Add the supernatant to 100ml of Renaturation Buffer R,
and stir gently at 4 degrees C. over night to renature the proteins.
The next day, the supernatant is clarified by centrifugation at 12,000 rpm for 30 min at 4 degrees C. The
supernatant contains highly purified expressed proteins.
Considerations
For plasmid #256 pGEX UTKMTB, step (7) of protocol:
Instead of suspending pellet in BUFFER D, 4M urea or 8M urea, inclusion bodies from #256 were resuspended
in a BUFFER containing 0.5% Deoxycholate; 0.2M Na CO3 pH 11.5, 5mM EDTA.
After step (3), 1X PIN and freshly prepared 1mM PMSF are added at each following s step.
BUFFER P Buffer W
BUFFER A
20mM Tris-HCl pH 7.5 PBS w/o Ca, Mg PBS w/o Ca, Mg
20% Sucrose 5mM EDTA 25% Sucrose
1mM EDTA 1X PIN, PMSF,--added fresh 5mM EDTA
1% Triton
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BUFFER R BUFFER D BUFFER U1
50Mm Tris-HCl pH 8.0 50mM Tris-Hcl pH 8.0 50mM Tris-HCl pH 8.0
1mM DTT 5M Guanidium HCl 4M Urea
20% Glycerol 5mM EDTA
BUFFER U2
50mM Tris HCl pH 8.0
8M Urea
Protocol specific Information
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Protocol for purification of His-tagged proteins synthesized using Expressway
Protocol for protein-protein interaction studies
Protocol for purification from E. Coli lysate
Protocol for purification from S. Cerevisiae
Protocol for purification from Pichia lysates
Protocol for purification from baculovirus lysates
Protocol for purification of His-tagged proteins synthesized in ExpressWay
(back to Table of Content)
(back to Protocol Specific Information)
Purification may be performed at 4掳C or room-temperature depending upon the sensitivity of the synthesized product.
1. Upon completion of incubation, remove desired portion of reaction for His-Tag purification to a clean
microcentrifuge tube. Add 4 volumes of Binding buffer and vortex briefly (Add 200 碌l for 50 碌l of reaction).
Centrifuge 5 minutes at 12,000 rpm.
2. Transfer the supernatant to a 2.0 ml tube containing 50 碌l pre-equilibrated Talon (or Ni-NTA) Resin.
3. Incubate with shaking or mixing 30-60 minutes.
4. Spin down resin for 2 minutes at 800xg (Do not spin any higher or the resin will collapse and recovery will be low).
Carefully remove supernatant.
5. Add 200碌l wash buffer and mix 5 minutes.
6. Spin down resin for 2 minutes at 800xg. Carefully remove supernatant.
7. Repeat steps 5 & 6.
8. Add 100 碌l Elution Buffer and mix 5 minutes.
9. Spin down resin for 2 minutes at 800xg. Carefully remove and SAVE supernatant.
10. Repeat steps 8 & 9.
Binding Buffer:
50 mM NaP04 pH 7.0
500 mM NaCl
6M Guanidine HCl (optional)**
Wash Buffer:
50 mM NaP04 pH 7.0
500 mM NaCl
15 鈥?25 mM Imidazole*
Elution Buffer:
50 mM NaP04 pH 7.0
500 mM NaCl
150 鈥?250 mM Imidazole*
**Depending on downstream applications, the purification may be performed under semi-denaturing
conditions, or native conditions. Under semi-denaturing conditions, the reaction is diluted in Denaturing
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Binding Buffer containing 6M Guanidine HCl, but washed and eluted with Native Buffers.
* The concentration of Imidazole is dependent upon the type of resin used. For Talon resins, a lower
amount is required (15 mM in the wash and 150 mM in the elution). For Ni-NTA or ProBond resins use
25 mM in the wash and 250 mM in the elution.
Protocol for Protein 鈥揚rotein Interaction Studies
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(back to Protocol Specific Information)
Science 2000 Aug 18; 289 (5482):1194-7
His6-Asef/APC bound to ProBond Resin (Invitrogen) was mixed with RhoA, Rac1, and GST-Cdc42 In Buffer A [50 mM
Hepes (pH 7.0), 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM DTT] phenylmethylsulphonyl fluoride (50 碌g/ml)
leupeptin (1 碌g/ml) aprotinin (1 碌g/ml)] containing 0.1% NP-40 for 1 hour at 4C and then washed extensively with buffer A.
Proteins adhering to the resin were analyzed by 13% SDS-PAGE and subjected to immunoblotting.
Protocol for Purification from E. Coli Lysate
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(back to Protocol Specific Information)
When purifying proteins from E coli lysates, one has to 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 His-tagged protein is low expressed or not expressed at all.
In its absence or low expression, SlyD will bind to the Ni+ column with great affinity.
Increase the purification stringency as discussed above to overcome SlyD binding.
Protocol for Purification from S. cerevisiae
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(back to Protocol Specific Information)
Over-expressed proteins should be purified as described in the Pichia manual below, i.e. K1740-01, see p. 41-42 under
Purification.. However, if the breaking buffer is used for S. cerevisiae, the EDTA should be omitted. In other words:
Breaking buffer if working for Pichia should contain ETDA, see p. 49 in K1740-01, but EDTA should be omitted if working
with S. cerevisiae (). The recipe for the breaking buffer is listed on p. 49 in K1740-01.
Protocol for Purification from Pichia Lysates
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(back to Protocol Specific Information)
Alcohol oxidase (AOX protein) is an octamer and has at least a few His stretches. Hence, AOX protein will bind to
Probond. In order to prevent co-elution, it is recommended to do an ion exchange purification prior to the Probond
purification. We routinely use HiQ column to which AOX will not bind. Users 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
High Q is a BioRad resin which is similar to Q Sepharose Fast Flow. Basically a quaternary ammonium resin for
anion exchange chromatography. Most proteins are anions at neutral pH and will stick well to a "Q" column at
neutral pH and low salt
In Pichia, over-expessed proteins should be purified according to the manual with cat K1740-01, see p. 3 and p. 41-
42 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 p. 49 of cat#
K1740-01.
Protocol for Purification from Baculovirus Lysates
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(back to Protocol Specific Information)
15
Purification of His-CAT from baculovirus infected Sf9 cells is typically done under various conditions and they are:
Denaturing purification whereby lysis was done in the presence of 6M guanidine or 8 M Urea.
Denaturing conditons gave a smaller yield but with reasonable purity. CAT was eluted between pH 5.3 and 4.0
Purification that applied different pH and imidazole concentrations
Samples are loaded at pH 7.8 and washed at pH 6.0. Washes at 20 mM, 50 mM 100mM and 200mM Imidazole
were performed. The 100mM imidazole elution had very pure CAT protein. The 20 mM had some contaminating
proteins and there was still some CAT in the 200mM. The pH elution was a little less clean; the CAT protein
already partially eluted in the wash fractions.
Native purification that was entirely based on an Imidazole step gradient. This method provided the best yield.
No CAT protein was seen at Imidazole concentrations < 100 mM.
PRODUCT DOCUMENTATION
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Brochures Citations Cell lines
COA FAQ Licensing
Manuals MSDS Newsletters
Vector Data
REFERENCES
(back to Table of Content)
Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press
In Science 2000 Aug 18; 289 (5482):1194-7
PRODUCT NAME AND CATALOG NUMBERS
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Name Size Part Number Catalog Number
Probond Nickel-Chelating 50 ml 460019 K850-01
Resin
Polypropylene Columns 3xc 50 ml 460019 K850-15
(empty)
Polyproylene Columns 50 450015 R640-50
(empty)
Probond Purification System 6 purifications 450055 contains buffers K850-01
460901 contains 12 resin
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Probond Purification with 6 purifications 450055 K851-01
Anti-Xpress antibody 460901
460528 has Xpress ab
Probond Purification with 6 purification 450055 K852-01
Anti-myc HRP antibody 460901
460709 has Myc-HRP ab
Probond Purification with 6 purifications 450055 K853-01
anti-His (C-term) HRP 460901
antibody 460707 has His-ab
Probond Purification with 6 purifications 450055 K854-01
anti V5-HRP Antibody 460901
460708 V5-HRP ab
Ni-NTA agarose 10 ml 600441 R901-01
25 ml 600442 R901-15
100 ml (4x 25 ml) 600442 (4x) R901-10
Ni-NTA purification system 6 purifications 450055 contains buffers K950-01
600441 12 ml
Ni-NTA purification system 6 purifications 450055 R951-01
with Anti-Xpress 600441
antibody 460528 has Xpress ab
Ni-NTA purification 6 purifications 450055 K952-01
systems with Anti-myc HRP 600441
antibody 460709 has Myc ab
Ni-NTA purification system 6 purifications 450055 K953-01
with Anti-His (C-Term)- 600441
HRP antibody 460707 has His ab
Ni-NTA Purification System 6 purifications 450055 K954-01
with Anti-V5-HRP antibody 600441
460708 has V5 ab
COMPONENTS
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Components of 450555 Size Composition
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5X Native Purification 125 ml 250 mM NaH2PO4
Buffer, pH 8.0 2.5 M NaCl
Guanidinium Lysis Buffer, 6 60 ml bottle 6 M Guanidine HCl
M, pH 7.8 500 mM NaCl
1.7 mMNaH2PO4.H2O
18.3 mM Na2HPO4.H2O
Denaturing Binding Buffer, 2 x 125 ml bottles 8 M Urea
pH 7.8 500 mM NaCl
1.7 mM NaH2PO4.H2O
18.3 mMNa2HPO4.H2O
Denaturing Wash Buffer, 2x 125ml bottles 8 M Urea
pH: 6.0 500 mM NaCl
17.5 mM NaH2PO4.H2O
2.5 mM Na2HPO4.H2O
Denaturing Elution Buffer, 1 x 60 ml bottle 8 M Urea
pH: 4.0 20 mM NaH2PO4
500 mM NaCl
Imidazole 1x 8 ml bottle 3 M Imidazole, pH 6.0
500 mM NaCl
17.5 mM NaH2PO4.H2O
2.5 mM Na2HPO4.H2O
ASSOCIATED PRODUCTS
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Gateway庐 Destination Vectors in www.invitrogen.com/gateway
Any expression, i.e. mammalian , insect, yeast, or E. coli destination vector that has either a C- or N-terminal His-tag
Topo cloning vectors in www.invitrogen.com/topo
Any topo expression, i.e. mammalian, insect, yeast or E. coli topo expression vector that has either a C- or N-terminal
His-Tag.Gateway庐 Technology Brochure
Invitrogen鈥檚 Traditional His-tagged Expression Vectors:
E. coli
PTrcHis V36020
PRSET V35120
PRSET/CFP V35220
PRSET/BFP V35420
PRSET/EmGFP V35320
Yeast 鈥? S. cerevisiae
PYes3/CT V825320
Pyes2/CT V825120
PYes2/NT V825220
Yeast- Pichia
PPicZ V19020
PpicZ alpha V19520
PGAPZ V20020, V20520
PGAPZ alpha V20020, V20520
Insect
PblueBacHis2 K87501, V37520
PBlueBac4.5/V5-His K207520
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PIZV5His K80001, K805001
Mammalian
PcDNA3.1- His V38520
PcDNA3.1(+)-Myc/His V80020
PcDNA3.1(-)-Myc/His V85520
PcDNA4-His V86220
PcDNA4-HisMax V86420
PcDNA4-V5His V86120
PcDNA4-MycHis V86220
PcDNA6-His V22220
PcDNA6-V5His V22020
PcDNA6-MycHis V22120
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