Table of Contents
About the Kits (2)
Description2
Components2
rEK Cleavage (3)
Small scale optimization3
Scale-up4
Monitoring cleavage4
rEK Capture (5)
Capture buffer considerations5
Monitoring rEK capture5
Inactivation of rEK6
References (6)
Copyright 1996–1998 by Novagen, Inc. All rights rerved.
EKapture™, S•Tag™, His•Bind®, pBAC™, the Novagen logo and name are trademarks of Novagen, Inc. CDP-Star is a trademark of and is manufactured by Tropix, Inc. SuperSignal is a trademark of and is manufactured by Pierce Chemical Co.
Recombinant enterokina is sold under patent licen from Genetics Institute, Inc. For Rearch U Only. Licens for commercial manufacture or u may be obtained directly from Genetics Institute, Inc., 87 Cambridge Park Drive, Cambridge, MA, 02140.
About the Kits
Enterokina Cleavage Capture Kit69067-3
Recombinant Enterokina69066-3
Description
The Enterokina Cleavage Capture Kit is designed for highly specific cleavage of fusion proteins
followed by the rapid, affinity-bad capture and removal of recombinant enterokina (rEK).
Following cleavage of the target protein, rEK is removed with > 99% efficiency from the reaction
by affinity capture on EKapture™ Agaro. Following capture of rEK, the EKapture Agaro is
removed by spin-filtration. Since the same buffer conditions are ud for both cleavage and
capture, no buffer changes are necessary.
Recombinant Enterokina is a highly purified preparation of the catalytic subunit of bovine
enterokina, which recognizes the identical cleavage site (AspAspAspAspLys↓) as the native
enzyme and has similar enzymatic activity. rEK exhibits superior rates of cleavage of fusion
proteins containing the recognition quence when compared to native enzyme (1). Novagen’s
rEK is purified to near homogeneity and, unlike some preparations of native bovine enterokina,
exhibits no condary cleavage arising from contaminating proteas. When analyzed by SDS-
PAGE on a 4–20% gradient gel under reducing conditions, rEK migrates as a single band with
apparent molecular weight of 26 kDa. One unit of rEK cleaves 50 µg fusion protein in 16 h at 20°C
in a buffer containing 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 2 mM CaCl
.
2
A Cleavage Control Protein is included for conducting control digests in parallel with
experimental samples, or to test cleavage under customized buffer conditions. The 48 kDa
心的成语control protein is cleaved by rEK into two proteolytic fragments of 32 kDa and 16 kDa which are
easily visualized by standard SDS-PAGE.
Components
澳凼大桥Enterokina Cleavage Capture Kit
The kit contains enough components to treat up to 2.5 mg of recombinant protein.
•50 U Recombinant Enterokina (in 20 mM Tris-HCl pH 7.4, 200 mM NaCl,
, 50% glycerol)
2 mM CaCl
2
•10 µg Cleavage Control Protein (in 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 20 mM EDTA,
50% glycerol)
• 2 ml 1X rEK Dilution/Storage Buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl,
2 mM CaCl
, 50% glycerol)
2
• 5 ml10X rEK Cleavage/Capture Buffer (200 mM Tris-HCl pH 7.4, 500 mM NaCl,
20 mM CaCl
)
2
• 1.5 ml EKapture Agaro (3 ml of a 50% slurry in phosphate buffer, pH 7.3, 0.5 M NaCl,
0.02% Thimerosal)
•10Spin Filters, 2-ml capacity
Storage: Store Spin Filters and EKapture Agaro at 4°C (Spin Filters can also be stored at room
temperature). Do not freeze EKapture Agaro. Store other kit components at –20°C.
Recombinant Enterokina Kit
The kit contains enough components to treat up to 2.5 mg of recombinant protein.
•50 U Recombinant Enterokina (in 20 mM Tris-HCl pH 7.4, 200 mM NaCl,
, 50% glycerol)
2 mM CaCl
关于母爱的
2
•10 µg Cleavage Control Protein (in 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 20 mM EDTA,
50% glycerol)
• 2 ml rEK Dilution/Storage Buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl,
2 mM CaCl
, 50% glycerol)
2
• 1 ml10X rEK Cleavage/Capture Buffer (200 mM Tris-HCl pH 7.4, 500 mM NaCl,
20 mM CaCl
)
2
Storage: Store kit components at –20°C.
Related products/available parately Size Cat. No.
Cleavage Control Protein10 µg69069
EKapture™ Agaro 1.5 ml69068-3
无人机发展S-protein HRP Conjugate50 µl69047-3
S-protein AP Conjugate50 µl69598-3
S-protein FITC Conjugate200 µl69060-3
S•Tag™ AP Western Blot Kit
25 blots69213-3
(colorimetric)
S•Tag HRP LumiBlot™ Kit25 blots69058-3
S•Tag AP LumiBlot Kit25 blots69099-3
CDP-Star™ Substrate40 ml69086-3
SuperSignal™ Substrate50 ml69059-3别人帮助我的作文
Spin Filters, 2-ml capacity pkg/1069072-3
Spin Filters, 5-ml capacity pkg/269074-3
rEK Cleavage
Recombinant Enterokina is a site-specific protea that exhibits very low non-specific cleavage
under many conditions. One unit of rEK is generally sufficient for cleavage of 50 µg target protein
in 1X rEK Cleavage/Capture Buffer at 20°C for 16 h. However, becau each target protein
prents the cleavage site somewhat differently, it is recommended to test veral rEK
concentrations, temperatures and/or incubation times to optimize specificity and efficiency of
cleavage. Note that excess rEK may result in unwanted proteolysis at condary sites. Incubation
temperatures ranging from 4°C to 37°C can be ud, although we recommend 20°C as the starting
point for most proteins. Testing can be carried out in small scale reactions, which can be scaled
up proportionately when optimal conditions are found.
Avoid the prence of rine protea inhibitors during cleavage (for example, rEK is effectively
inhibited by 1 mM PMSF).
Small scale optimization
For small scale digestions the rEK can be diluted in rEK Dilution/Storage Buffer. The dilutions
can be stored in this buffer at –20°C for veral weeks; however, to avoid loss of activity we do
not recommend extended storage of dilutions. The following protocol is an example of a simple
optimization experiment designed to estimate the appropriate range of enzyme:target protein.
1.Make 3 rial dilutions of rEK in rEK Dilution/Storage Buffer to produce solutions having 0.1,
0.2, and 0.5 U enzyme per µl.
2.Asmble the following components in a ries of 4 labeled tubes.
5 µl10X rEK Cleavage/Capture Buffer
10 µg target protein
1 µl Diluted rEK (each tube receives 1 µl of a
different enzyme dilution. The fourth tube
receives 1 µl Dilution/Storage Buffer only
as a negative control)
x µl deionized water
50 µl total volume
3.Incubate the reactions at room temperature (20–21°C), taking 10 µl aliquots into 10 µl 2X SDS
sample buffer after 2, 4, 8 and 16 h.
4.Determine the extent of cleavage of the samples by SDS-PAGE analysis.
Scale-up
When a satisfactory condition is found, scale up the reaction proportionately. Note that if the reaction volume is kept in proportion from the above small scale example, a relatively large volume (5 ml) would be ud for a 1 mg digestion. If desired, another preliminary experiment can be performed in which the reaction volume is varied while keeping the enzyme:target protein ratio and incubation con
ditions constant, which will determine appropriate adjustments needed for higher concentrations of enzyme and target protein.
The highest reaction rates and best cleavage efficiencies are demonstrated when the target protein is maintained at a concentration of 2.5 µM or above. Bad on this target concentration, 10 µg of a target protein with a molecular weight of 50 kDa should be cleaved in a total volume of 80 µl or less. If the target protein is too dilute, pilot digestions of the control protein can be performed to determine the amount of rEK necessary to achieve cleavage, or the target protein can be concentrated. Examples of methods for concentrating proteins include:
1.Place the sample in dialysis tubing with an exclusion limit of 3500 MW and concentrate by水瓶男的性格特征
sprinkling solid polyethylene glycol (15,000–20,000 molecular weight) or Sephadex G50
(Pharmacia) on the dialysis tubing. Leave the solid in contact with the tubing until the desired volume is reached, replacing it with fresh solid as necessary.
2.U plastic disposable micro-concentrator units (e.g., Centricon, Amicon) as directed by the
manufacturer to both desalt and concentrate the sample by ultrafiltration.
Notes:
a)The optimal enzyme specificity is achieved using the lowest amount of protea necessary to
原物璧还
achieve complete cleavage.
b)Salt nsitivity: rEK is inhibited by imidazole or NaCl concentrations above 250 mM.
c)Denaturants or chaotropes: activity of the enzyme in solutions containing urea or guanidine
should be confirmed with pilot digestions. rEK is especially nsitive to urea concentrations above 2 M.
d)Detergents: when digests are performed with as little as 0.0625% (w/v) SDS, significant
condary cleavage is en. For this reason, it is recommended that SDS be avoided. rEK will tolerate Triton X-100 at concentrations of 1% without affecting specificity or activity.
e)Incubation temperature: rEK has higher activity at 21°C than at 37°C.
心理委员的职责Monitoring cleavage
Cleavage can be easily monitored by including a parallel reaction using the supplied Cleavage Control Protein in the same buffer system as the target protein. The Cleavage Control Protein is converted from a single 48 kDa band to two bands of 32 kDa and 16 kDa following rEK cleavage. Inclusion of a control digest enables confirmation of enzyme activity and cleavage specificity, which is especially important when cleavage conditions have been modified. Whereas sufficient control protein is provided to allow Coomassie detection of cleavage products, smaller scale reactions (< 0.5 µg) can be monitored by Western blotting. The Cleavage Control Protein has an S•Tag peptide on the amino-terminal side of the rEK recognition quence. The 16 kDa cleavage product can be detected using either S-protein AP Conjugate or S-protein HRP Conjugate. High nsitivity chemiluminescent detection of S•Tag fusion proteins can be performed using the S•Tag AP or HRP LumiBlot Kits. Also included in the S•Tag LumiBlot Kits is a convenient t of Perfect Protein Western Blot Markers to confirm the molecular weight of cleavage reaction products.
rEK cleavage can also be monitored by testing for the removal of peptides from fusion proteins upstream of the cleavage site. For example, S•Tag fusion proteins can be blotted with and without rEK treatment and detected with any of the S•Tag Western Blot Kits. Successful cleavage results in removal of the S•Tag peptide and no band will be evident at the size corresponding to the target prot
ein. A similar strategy can be ud with T7•Tag®, CBD•Tag™, or other peptides that may be cleaved from fusion proteins and for which a detection reagent is available.
rEK Capture
After the cleavage reaction, rEK can be effectively removed with EKapture Agaro (supplied in the rEK Cleavage/Capture Kit). Following cleavage of the target protein, the rEK is bound
batchwi to EKapture Agaro and the target protein recovered by spin-filtration. When using 1X rEK Cleavage/Capture Buffer, a ratio of 50 µl ttled resin (100 µl of the 50% slurry) per 2 units of enzyme will bind > 99% of the enzymatic activity in a 5 min incubation. Recovery of cleaved target protein is simplified by u of supplied Spin Filters, which enable efficient paration of the liquid pha of the reaction from the EKapture Agaro. For demanding applications where more stringent removal of rEK is required, two or more capture steps are recommended.
Alternatively, any remaining rEK can be inactivated as described later in this ction.
1. Determine the required amount of EKapture Agaro necessary to capture the rEK prent in the cleavage reaction. The EKapture Agaro has sufficient capacity to bind 2 units rEK per 50µl bed vol
ume (100 µl slurry) in 1X rEK Cleavage/Capture Buffer. (If using a buffer other than the supplied rEK Cleavage/Capture Buffer, e “Capture buffer considerations” below). We
recommend using a minimum of 25 µl EKapture Agaro slurry becau smaller resin volumes are difficult to manipulate.
Caution:The EKapture Agaro is stored in a buffer containing 0.02% Thimerosal and should be handled with
caution. Wear gloves and appropriate laboratory clothing.
2. Prepare 1X rEK Cleavage/Capture Buffer by diluting the supplied 10X stock with sterile deionized water. You will need an amount of buffer corresponding to approximately 11 bed volumes of EKapture Agaro (as determined in Step 1).
3. Mix the EKapture Agaro (supplied as a 50% slurry) by inversion until fully resuspended.Using a wide mouth pipette, transfer the required amount of slurry into a clean centrifuge tube (transfer twice the required bed volume to account for the buffer).
4. Centrifuge at 1000 × g for 5 min and carefully remove and discard the supernatant.
5. Resuspend the agaro in ten bed volumes of 1X rEK Cleavage/Capture Buffer, centrifuge
again, and remove and discard the supernatant.
6. Add one bed volume of 1X rEK Cleavage/Capture Buffer and fully resuspend. The EKapture Agaro is now equilibrated and ready to u.
7. Transfer the prepared EKapture Agaro from Step 6 to the sample cup of a 2-ml Spin Filter (included with the kit). Add the entire volume of the cleavage reaction to the prepared
EKapture Agaro. For total volumes in excess of 2 ml, u a centrifuge tube or larger Spin
Filter. Mix gently to resuspend the agaro. Do not vortex.
8. Incubate at room temperature for 5 min.
9. Centrifuge the reaction at 1000 × g for 5 min to remove the EKapture Agaro. Bound rEK is retained in the sample cup, and the cleaved target protein flows into the filtrate tube during
centrifugation.
Capture buffer considerations
The binding of rEK to EKapture Agaro is nsitive to some buffer conditions in which rEK is fully active. If buffers other than the rEK Cleavage/Capture Buffer are required to maintain target protein solubility or activity, it is important to consider the impact such changes have on the
affinity capture step. For example, the prence of 2M urea will reduce the capture efficiency of EKapture Agaro approximately 60%. Likewi, the prence of 0.25X His•Bind ® Elution Buffer or other salts will reduce capture by 20-50%. EKapture binding is unaffected by DTT at
concentrations up to 100 mM, and Triton X-100 up to 1%. The supplied rEK Cleavage/Capture Buffer is compatible with both cleavage and capture steps without any loss of efficiency of either step.
Monitoring rEK capture
To determine capture efficiency, perform pilot capture reactions under the modified buffer
conditions and assay the unretained fractions for rEK activity, A simple, rapid fluorometric
For maximum capture
efficiency, EKapture
Agaro should be pre-
equilibrated in 1X rEK
Cleavage/Capture buffer
prior to u (Steps 2–6).
