ANTI-FLAG®M2 Affinity Gel
Catalog Number A2220
Storage Temperature –20 °C
TECHNICAL BULLETIN
Product Description
ANTI-FLAG®M2 affinity gel is a purified murine IgG1 monoclonal antibody covalently attached to agaro by hydrazide linkage. It is uful for purification or immunoprecipitation of FLAG®fusion proteins.
ANTI-FLAG M2 binding to the FLAG peptide is not calcium dependent.
Binding Specificity:
FLAG octapeptide (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) at N-terminal, Met-N-terminal, C-terminal, and internal locations of a fusion protein.
Reagent
ANTI-FLAG M2 affinity gel is supplied as a 50% suspension in 50% glycerol with 10mM sodium phosphate and 150mM sodium chloride, pH7.4, containing 0.02% (w/v) sodium azide (PBS/A).
Equipment and Reagents Required but Not Provided
packages• Cells expressing FLAG fusion protein
• Lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl,1mM EDTA, and 1% TRITON®X-100),
CelLytic™M (Catalog Number C2978), or
CelLytic B (Catalog Number B7435, B7310, or
C8740)
• Appropriate centrifuge
• Appropriate column or centrifuge tubes
• Sodium chloride, Catalog Number S3014
• Trizma®ba, Catalog Number T6066
• Protea inhibitor cocktail for u with mammalian cells and tissue extracts, Catalog Number P8340
Precautions and Disclaimer
This product is for R&D u only, not for drug, houhold, or other us. Plea consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.Storage/Stability
ANTI-FLAG M2 affinity gel should be stored in 50% glycerol at –20°C for maximum stability. The unopened product is stable for one year when stored as indicated. After u, the resin should be cleaned and stored in 50%glycerol with TBS or PBS buffer containing 0.02% sodium azide to protect the product. Do not freeze in the abnce of glycerol.
Procedure
Note: It is recommended that the entire technical bulletin be read before u, especially the Reagent Compatibility Table.
Part I. Cell Lysate Preparation
The rearcher must empirically determine the most suitable procedure. Typical methods for purifying FLAG fusion proteins from crude E. coli extracts are provided. It is recommended that the CelLytic B Lysis Reagents (Catalog Numbers B7435, B7310, or C8740) or CelLytic B Plus Kit (Catalog Numbers CB0050 or CB0500) products be ud for bacterial cell lysis. CelLytic M can be ud for mammalian cells.
A.Recommended procedure for E. coli using
CelLytic Lysis Reagents
1.Grow the cells (∼1liter or less) under
conditions that induce production of FLAG
fusion proteins.
2.Harvest the cells by centrifugation at 5,000×g
for 30 minutes at 2−8 °C.
3.Decant the medium from the cell paste.
4.Freeze the cell paste using a dry ice/ethanol
bath or at –20 °C in a freezer. Cell lysis is
enhanced during the slow freezing.
5.Ly the frozen cells with 10 ml of CelLytic B
(Catalog Number B7435) per g of frozen cell
paste or 5 ml of CelLytic B, 2×concentrate
(Catalog Number B7310) per g of frozen cell
paste.
2
6.Resuspend the cells in the CelLytic B reagent
with a pipette. Mix vigorously on a stir plate for
15minutes to fully extract the protein.
7.Remove the cell debris by centrifuging for
15minutes at 21,000 ×g.
8.After centrifugation, decant the supernatant
into a fresh container and dispo of the cell
pellet. The solution should be clear with no
insoluble particles.
B.Recommended procedure for mammalian cells
For a 70–90% confluent 100 mm dish (106–107cells), u 1 ml of lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl,1 mM EDTA, and 1% TRITON X-100). If the expression level of the FLAG fusion protein is relatively low, ly the cells with a reduced volume of lysis buffer. It is highly recommended to add a protea inhibitor cocktail (Catalog Number P8340) to the lysis buffer (10 µl per 1 ml of lysis buffer), especially if the lysate is to be stored for further u.
1.Wash adherent or suspension cells as
appropriate:
Adherent Cells-Remove the growth medium
from the cells to be analyzed. Rin the cells
twice with PBS (10mM phosphate, 2.7mM
potassium chloride, and 137 mM sodium
chloride, pH 7.4, at 25°C) buffer, being careful
not to dislodge any of the cells. Discard the
制版师
PBS. Add lysis buffer (106–107cells/ml).
Cells in Suspension-Collect the cells into an
appropriate conical centrifuge tube. Centrifuge
for 5minutes at 420 ×g. Decant the
supernatant and discard. Wash the cells twice
by resuspending the cell pellet with PBS and
centrifuge for 5minutes at 420×g. Decant the
supernatant and discard. Resuspend the cell
judger
pellet in lysis buffer (106–107cells/ml).
2.Incubate the cells for 15–30 minutes on a
shaker.
3.For adherent cells only, scrape and collect the
cells. For cells in suspension, proceed to
step4.
4.Centrifuge the cell lysate for 10 minutes at
12,000×g.
5.Transfer the supernatant to a chilled test tube.
For immediate u, keep on ice. If the
supernatant is not to be ud immediately,
store it at –70°C.Part II.Resin Preparation
The ANTI-FLAG M2 affinity resin is stored in 50% glycerol with buffer. The glycerol must be removed just prior to u and the resin equilibrated with buffer. The equilibration can be done at room temperature or at
杂志社英文2–8 °C. Remove only the amount of resin necessary for the purification. Thoroughly resuspend the resin. The matrix may then be poured into a clean chromatography column using standard techniques. Do not allow the resin to remain in TBS buffer for extended periods of time (>24 hours) unless an antimicrobial agent (e.g., 0.02% sodium azide) is added to the buffer.
1.Place the empty chromatography column on a firm
support.
2.Rin the empty column twice with TBS (50mM
Tris HCl, with 150mM NaCl, pH7.4) or another
appropriate buffer. Allow the buffer to drain from
the column and leave residual TBS in the column
to aid in packing the ANTI-FLAG M2 affinity gel. 3.Thoroughly suspend the resin by gentle inversion.
Make sure the bottle of ANTI-FLAG M2 affinity gel is a uniform suspension of gel beads. Remove an appropriate aliquot for u.
4.Immediately transfer the suspension to the
column.
5.Allow the gel bed to drain and rin the pipette
ud for the resin aliquot with TBS. The 50%
glycerol buffer will flow slowly and the flow rate will increa during the equilibration.
6.Add the rin to the top of the column and allow to
drain again. The gel bed will not form channels
when excess solution is drained under normal
circumstances, but do not let the gel bed run dry.
7.Wash the gel by loading three quential column
volumes of 0.1 M glycine HCl, pH3.5. Avoid
disturbing the gel bed while loading. Let each
aliquot drain completely before adding the next.
Do not leave the column in glycine HCl for
longer than 20minutes.
8.Wash the resin with 5 column volumes of TBS to
equilibrate the resin for u. Do not let the bed run dry. Allow a small amount of buffer to remain on
the top of the column.
Part III.Binding Procedures
For purification of FLAG fusion proteins, the resin can be ud in either a column or batch format. A column using 1–3 ml of resin will work well if the volume of cell lysate to be loaded is only ∼100 ml. For larger volumes of lysate, the batch format is recommended to quickly capture the target protein from a large volume of extract. If a small sample (1–2ml of cell lysate) is being purified, the FLAG fusion protein can be immunoprecipitated.
3
Pre-equilibrate the column and buffers, and perform the purification at room temperature. If there is a problem with proteas, perform column chromatography at 2–8 °C or add a protea inhibitor cockt
ail to the elution solution. Cellular debris and particulate matter can clog the column and must be removed prior to purification. Highly viscous samples containing chromosomal DNA or RNA can also clog the column and should be treated with an endonuclea such as Benzona®(Catalog Number E1014) to reduce viscosity. FLAG-BAP™positive control proteins can be ud to verify the functionality of the gel.
童话mp3Note: The ANTI-FLAG M2 affinity gel is resistant to many detergents. Do not u reagents that are harmful or potentially harmful to antibodies or proteins in general. See the Reagent Compatibility Table for more detail.
A.Binding FLAG Fusion Proteins to the Column
1.Proper binding of FLAG fusion proteins to the
ANTI-FLAG M2 affinity column requires
0.15M sodium chloride and neutral pH.
2.Load the sample onto the column under
gravity flow. Fill the column completely veral
times or attach a column rervoir prior to
loading for larger volumes. Depending upon
the protein and flow rate, all of the antigen
may not bind. Multiple pass over the column
will improve the binding efficiency.
3.Wash the column with 10–20 column volumes
of TBS. This should remove any proteins that
are not bound to the M2 antibody. Allow the
column to drain completely.
B.Select one of the two following procedures for
elution.
1.Elution of FLAG Fusion Proteins by Acid
Elution with Glycine –Elute the bound FLAG
fusion protein from the column with six 1ml
aliquots of 0.1M glycine HCl, pH3.5, into vials
containing 15–25µl of 1M Tris, pH8.0. Do not
leave the column in the glycine HCl solution
for longer than 20minutes. Re-equilibrate to
neutral pH as soon as possible after elution.
Or
2.Elution of FLAG Fusion Proteins by
Competition with FLAG Peptide –Elute the
hero的意思
bound FLAG fusion protein by competitive
elution with five one-column volumes of a
solution containing 100µg/ml FLAG peptide
(Catalog Number F3290) in TBS.C.Recycling the Column
It is recommended that the column be regenerated immediately after u by washing with three
column volumes of 0.1 M glycine HCI, pH3.5. The column should be immediately re-equilibrated in
TBS until the effluent is at neutral pH.
D.Storing the Column
Wash the column with ten column volumes of 50% glycerol with TBS or PBS buffer containing 0.02% sodium azide, then add another 5 ml of buffered
glycerol containing 0.02% sodium azide and store at 2–8 °C or –20 °C without draining. When E. coli periplasmic extracts are applied to the column, it
may be reud up to 20times without loss of
binding capacity. When E. coli crude cell extracts
are applied to the column, it may be reud
3 times before loss of binding capacity is
obrved. The number of cycles obrved will be
dependent on variables such as sample condition, proteas etc.
2.Batch Absorption of FLAG Fusion Proteins using ANTI-FLAG M2 Affinity Gel
This method provides a quick and efficient way to purify FLAG fusion proteins from a dilute solution. It eliminates the time-consuming column chroma-tography step of placing a large volume of solution through a small amount of resin.
A.Adjust the pH of the protein extract to between
pH 7–8. It is also uful to have a salt (sodium or
potassium chloride) concentration of at least
0.15 M to reduce the number of proteins
nonspecifically binding to the resin.
B.The FLAG fusion protein extract must be clarified
to remove any insoluble material. A large amount
of insoluble material may require centrifugation
(10,000–20,000×g for 15minutes) for removal.
The protein extract should also be filtered with a
0.45 or 0.22µm filter to remove any remaining
cells and particulates that may clog the column or filter during collection of the resin in step F.
C.The ANTI-FLAG M2 affinity gel must be
equilibrated before u. See Procedure, Resin
Preparation ction.
D.Resuspend the resin in TBS and add to the protein
extract.
abloy
E.Incubate the protein extract with the ANTI-FLAG
M2 affinity gel for ∼1hour with gentle mixing to
capture the FLAG fusion proteins. Mixing should
be done on either an overhead mixing device or a platform shaker.
4
Do not u a magnetic stirring system becau this will destroy the resin beads.This step can
be done at 2–8 °C or at room temperature. This
incubation can go for as short as 30 minutes up to veral hours. If the incubation is longer than
3hours, protea inhibitors and antimicrobial
substances should be added to prevent microbial
growth and/or proteolysis.
工业工程就业方向F.Once the binding step is complete, collect the
resin from the container. The resin can be
collected by centrifugation (1,000 ×g for
5minutes) or by filtration, either in an empty
column or on a Buchner funnel.
G.Wash the resin with TBS to remove all of the
nonspecific proteins. This may be done in the
column format by passing fresh buffer through the column until no more protein elutes off. The新东方六级作文预测
protein being eluted from the resin can be
monitored by measuring the absorbance of the
eluant at 280nm. Continue washing the resin until the absorbance difference of the wash solution
coming off the column is less than 0.05 versus a
wash solution blank.
H.The FLAG proteins can be eluted from the resin
either by low pH or by competition with the FLAG
peptide. Follow the elution steps under Column
Chromatography, ction B.
I.The resin can be recycled and stored as described
under Column Chromatography, ctions C and D.
3. Immunoprecipitation of FLAG Fusion Proteins
This method is recommended for the purification of small amounts of FLAG fusion proteins.
Note: For antigens and protein:protein complexes requiring a special lysis buffer compod of a different percentage of a detergent, it is recommended to pretest the resin before u. The ANTI-FLAG M2 affinity gel is resistant to the many detergents such as 5.0% TWEEN®20, 5.0% TRITON X-100, 0.1% IGEPAL®CA-630, 0.1% CHAPS, and 0.2% digitonin. It can also be ud with 1.0M NaCl or 1.0 M urea. See the Reagent Compatibility Table for additional chemicals.
Perform all steps at 2–8 °C, unless the procedure specifies otherwi. U pre-cooled lysis and wash buffers and equipment. Do not pre-cool the cell lysate and elution buffers. Perform all centrifugations at
2–8 °C with pre-cooled rotors.A.FLAG Fusion Protein Immunoprecipitation
The procedure described below is an example of a single immunoprecipitation reaction. For multiple immunoprecipitation reactions, calculate the volume of reagents needed according to the number of
samples to be procesd. For easy performance of immunoprecipitation reactions, it is recommended to u 40µl of gel suspension per reaction (∼20µl of packed gel volume). Smaller amounts of resin (∼10µl of packed gel volume, which binds >1µg FLAG fusion protein) can be ud.
Note: Two control reactions are recommended for the procedure. The first control is immunoprecipitation with FLAG-BAP fusion protein (positive control) and the cond is a reagent blank with no protein (negative control).
1.Thoroughly suspend the ANTI-FLAG M2 affinity
gel in the vial, in order to make a uniform
suspension of the resin. The ratio of suspension to packed gel volume should be 2:1. Immediately
transfer 40 µl of the gel suspension to a fresh test tube. For resin transfer, u a clean, plastic pipette tip with the end enlarged to allow the resin to be
transferred.
2.Centrifuge the resin at 5,000–8,200×g for
30conds. In order to let the resin ttle in the
tube, wait for 1–2 minutes before handling the
samples. Remove the supernatant with a narrow-
end pipette tip or a Hamilton®syringe, being
careful not to transfer any resin. Narrow-end
pipette tips can be made using forceps to pinch
the opening of a plastic pipette tip until it is
partially clod.
3.Wash the packed gel twice with 0.5ml of TBS. Be
sure that most of the wash buffer is removed and
no resin is discarded. In ca of numerous
immunoprecipitation samples, wash the resin
needed for all samples together. After washing,
divide the resin according to the number of
samples tested. Each wash should be performed
with TBS at a volume equal to 20times the total
packed gel volume.
4.Optional Step -In order to remove any traces of
an unbound ANTI-FLAG antibody from the resin
suspension, wash the resin with 0.5 ml of 0.1 M
glycine HCl, pH 3.5, before continuing with the
binding step. Do not leave the resin in glycine
HCl for longer than 20 minutes.Discard the
supernatant immediately, being careful to remove all supernatant from the resin, and follow with
three washes consisting of 0.5 ml of TBS each.
5.Add 200–1,000 µl of cell lysate to the washed
resin. If necessary, bring the final volume to 1ml
by adding lysis buffer (50 mM Tris HCl, pH 7.4,
150mM NaCl,1 mM EDTA, 1% TRITON X-100).
5
Three elution methods are recommended according to protein characteristics or further usage:
1.Protein elution under native conditions by
competition with 3X FLAG peptide. The elution
efficiency is very high using this method.
2.Elution under acidic conditions with 0.1 M glycine
HCl, pH3.5. This is a fast and efficient elution
method. Equilibration of the eluted protein with
wash buffer may help prerve its activity.
3.Elution with sample buffer for gel electrophoresis
and immunoblotting.
1. Elution with3X FLAG peptide
a.Prepare 3X FLAG elution solution. Dissolve
3X FLAG peptide (Catalog Number F4799) in
0.5 M Tris HCl, pH 7.5, with 1M NaCl at a
concentration of 25 µg/µl. Dilute 5-fold with
water to prepare a 3X FLAG stock solution
containing 5µg/µl of 3X FLAG peptide. For
elution, add 3µl of 5µg/µl of3X FLAG peptide
stock solution to 100µl of TBS (150 ng/µl final
concentration).
b.Add 100 µl of 3X FLAG elution solution to
each sample and control resin.
c.Incubate the samples and controls with gentle
shaking for 30 minutes at 2–8 °C.
d.Centrifuge the resin for 30 conds at
5,000–8,200×g. Transfer the supernatants to
fresh test tubes using a Hamilton syringe or
equivalent device. Be careful not to transfer
any resin.
e.For immediate u, store the supernatants at
2–8°C. Store at –20 °C for long term storage.
2. Elution with 0.1M glycine HCl, pH
3.5
The procedure should be performed at room temperature.Do not leave the resin in this buffer more than 20minutes.
a.Add 100 µl of 0.1M glycine HCl, pH3.5, buffer
to each sample and control resin.
b.Incubate the samples and controls with gentle
shaking for 5minutes at room temperature.
c.Centrifuge the resin for 30 conds at
5,000–8,200×g. Transfer the supernatants to
fresh test tubes containing 10µl of 0.5M Tris
HCl, pH 7.4, with 1.5 M NaCl, using a
Hamilton syringe or equivalent device. Be
careful not to transfer any resin.
d.For immediate u, store the supernatant at
2–8°C. Store at –20 °C for long term storage.
3. Elution with SDS-PAGE Sample Buffer
The procedure should be preformed at room temperature. Sample buffer should be at room temperature before u. In order to minimize the denaturation and elution of the antibody, no reducing agent (2-mercaptoethanol or DTT) should be included in the sample buffer. The addition of reducing agents will result in the dissociation of the heavy and light chains of the immobilized M2 antibody (25 and 50kDa bands). If reducing conditions are absolutely necessary, a reducing agent may be added. The final concentration of 2-mercaptoethanol or DTT in the
1×sample buffer (62.5 mM Tris HCl, pH6.8, with 2% SDS, 10% (v/v) glycerol, and 0.002% bromphenol blue) should be 5% or 50mM, respectively.
Note: SDS in the sample buffer will denature the M2 antibody, and the ANTI-FLAG M2 affinity gel cannot be reud after treatment with the SDS-PAGE sample buffer.
a.Add 20 µl of2×sample buffer (125 mM Tris
HCl, pH 6.8, with 4% SDS, 20% (v/v) glycerol,
and 0.004% bromphenol blue) to each sample
and control.
b.Boil the samples and controls for 3minutes.
c.Centrifuge the samples and controls at
5,000–8,200×g for 30conds to pellet any
undissolved agaro. Transfer the
supernatants to fresh test tubes with a
Hamilton syringe or a narrow-end Pasteur
pipette. The samples and controls are ready
for loading on SDS-PAGE and immunoblotting
using ANTI-FLAG or specific antibodies
苏州软件测试培训against the fusion protein.
