ANTI-FLAG M2 Affinity Gel (A2220) - Technical ... - Sigma-Aldrich

ANTI-FLAG ® M2 Affinity Gel 

Catalog Number A2220 

Storage Temperature –20 °C 

Product Description 

ANTI-FLAG ® M2 affinity gel is a purified murine IgG1 

monoclonal antibody covalently attached to agarose 

by hydrazide linkage. It is useful 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 10 mM sodium 

phosphate and 150 mM sodium chloride, pH 7.4, 

containing 0.02% (w/v) sodium azide (PBS/A). 

Equipment and Reagents Required but Not 

Provided 

• Cells expressing FLAG fusion protein 

• Lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM 

NaCl, 1 mM 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 ® base, Catalog Number T6066 

• Protease inhibitor cocktail for use with mammalian 

cells and tissue extracts, Catalog Number P8340 

Precautions and Disclaimer 

This product is for R&D use only, not for drug, 

household, or other uses. Please consult the Material 

Safety Data Sheet for information regarding hazards 

and safe handling practices. 

TECHNICAL BULLETIN 

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 use, 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 absence of glycerol. 

Procedure 

Note: It is recommended that the entire technical 

bulletin be read before use, especially the Reagent 

Compatibility Table. 

Part I. Cell Lysate Preparation 

The researcher 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 used for bacterial cell lysis. 

CelLytic M can be used for mammalian cells. 

A. Recommended procedure for E. coli using 

CelLytic Lysis Reagents 

1. Grow the cells (∼1 liter 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. Lyse 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 

15 minutes to fully extract the protein. 

7. Remove the cell debris by centrifuging for 

15 minutes at 21,000 × g. 

8. After centrifugation, decant the supernatant 

into a fresh container and dispose 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 (10 6 –10 7 cells), 

use 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, lyse the cells with a reduced volume of 

lysis buffer. It is highly recommended to add a 

protease 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 use. 

1. Wash adherent or suspension cells as 

appropriate: 

Adherent Cells - Remove the growth medium 

from the cells to be analyzed. Rinse the cells 

twice with PBS (10 mM phosphate, 2.7 mM 

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 

PBS. Add lysis buffer (10 6 –10 7 cells/ml). 

Cells in Suspension - Collect the cells into an 

appropriate conical centrifuge tube. Centrifuge 

for 5 minutes at 420 × g. Decant the 

supernatant and discard. Wash the cells twice 

by resuspending the cell pellet with PBS and 

centrifuge for 5 minutes at 420 × g. Decant the 

supernatant and discard. Resuspend the cell 

pellet in lysis buffer (10 6 –10 7 cells/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 

step 4. 

4. Centrifuge the cell lysate for 10 minutes at 

12,000 × g. 

5. Transfer the supernatant to a chilled test tube. 

For immediate use, keep on ice. If the 

supernatant is not to be used 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 use and the resin equilibrated with buffer. The 

equilibration can be done at room temperature or at 

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. Rinse the empty column twice with TBS (50 mM 

Tris HCl, with 150 mM NaCl, pH 7.4) 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 use. 

4. Immediately transfer the suspension to the 

column. 

5. Allow the gel bed to drain and rinse the pipette 

used for the resin aliquot with TBS. The 50% 

glycerol buffer will flow slowly and the flow rate will 

increase during the equilibration. 

6. Add the rinse 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 sequential column 

volumes of 0.1 M glycine HCl, pH 3.5. 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 20 minutes. 

8. Wash the resin with 5 column volumes of TBS to 

equilibrate the resin for use. 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 used 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 (1–2 ml of cell lysate) is 

being purified, the FLAG fusion protein can be 

immunoprecipitated.

1. Column Chromatography 

Pre-equilibrate the column and buffers, and perform 

the purification at room temperature. If there is a 

problem with proteases, perform column 

chromatography at 2–8 °C or add a protease inhibitor 

cocktail 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 

endonuclease such as Benzonase ® (Catalog Number 

E1014) to reduce viscosity. FLAG-BAP positive 

control proteins can be used to verify the functionality 

of the gel. 

Note: The ANTI-FLAG M2 affinity gel is resistant to 

many detergents. Do not use 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 

0.15 M sodium chloride and neutral pH. 

2. Load the sample onto the column under 

gravity flow. Fill the column completely several 

times or attach a column reservoir prior to 

loading for larger volumes. Depending upon 

the protein and flow rate, all of the antigen 

may not bind. Multiple passes 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 1 ml 

aliquots of 0.1 M glycine HCl, pH 3.5, into vials 

containing 15–25 μl of 1 M Tris, pH 8.0. Do not 

leave the column in the glycine HCl solution 

for longer than 20 minutes. 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 

bound FLAG fusion protein by competitive 

elution with five one-column volumes of a 

solution containing 100 μg/ml FLAG peptide 

(Catalog Number F3290) in TBS. 

C. Recycling the Column 

It is recommended that the column be regenerated 

immediately after use by washing with three 

column volumes of 0.1 M glycine HCI, pH 3.5. 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 reused up to 20 times without loss of 

binding capacity. When E. coli crude cell extracts 

are applied to the column, it may be reused 

3 times before loss of binding capacity is 

observed. The number of cycles observed will be 

dependent on variables such as sample condition, 

proteases 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 chromatography 

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 useful 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 15 minutes) for removal. 

The protein extract should also be filtered with a 

0.45 or 0.22 μm 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 use. See Procedure, Resin 

Preparation section. 

D. Resuspend the resin in TBS and add to the protein 

extract. 

E. Incubate the protein extract with the ANTI-FLAG 

M2 affinity gel for ∼1 hour with gentle mixing to 

capture the FLAG fusion proteins. Mixing should 

be done on either an overhead mixing device or a 

platform shaker. 

3

4 

Do not use a magnetic stirring system because 

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 

several hours. If the incubation is longer than 

3 hours, protease inhibitors and antimicrobial 

substances should be added to prevent microbial 

growth and/or proteolysis. 

F. Once the binding step is complete, collect the 

resin from the container. The resin can be 

collected by centrifugation (1,000 × g for 

5 minutes) 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 

protein being eluted from the resin can be 

monitored by measuring the absorbance of the 

eluant at 280 nm. 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, section B. 

I. The resin can be recycled and stored as described 

under Column Chromatography, sections 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 composed of a different 

percentage of a detergent, it is recommended to 

pretest the resin before use. 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 used with 1.0 M 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 otherwise. Use 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 processed. For easy performance of 

immunoprecipitation reactions, it is recommended to 

use 40 μl of gel suspension per reaction (∼20 μl of 

packed gel volume). Smaller amounts of resin (∼10 μl 

of packed gel volume, which binds >1 μg FLAG fusion 

protein) can be used. 

Note: Two control reactions are recommended for the 

procedure. The first control is immunoprecipitation with 

FLAG-BAP fusion protein (positive control) and the 

second 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, use 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 

30 seconds. In order to let the resin settle in the 

tube, wait for 1–2 minutes before handling the 

samples. Remove the supernatant with a narrowend 

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 closed. 

3. Wash the packed gel twice with 0.5 ml of TBS. Be 

sure that most of the wash buffer is removed and 

no resin is discarded. In case 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 20 times 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 1 ml 

by adding lysis buffer (50 mM Tris HCl, pH 7.4, 

150 mM NaCl, 1 mM EDTA, 1% TRITON X-100).

The volume of cell lysate to be used depends on 

the expression level of FLAG fusion protein in the 

transfected cells. For the positive control, add 1 ml 

of TBS and 4 μl of 50 ng/μl FLAG-BAP fusion 

protein (∼200 ng) to the washed resin. For the 

negative control, add only 1 ml of lysis buffer with 

no protein. The amount of FLAG-BAP fusion 

protein to be precipitated depends on the 

detection method. 200 ng of protein is sufficient for 

an activity assay or for an immunoblot analysis. 

For SDS-PAGE analysis with Coomassie ® blue or 

silver staining, use 1 μg of FLAG-BAP fusion 

protein. 

6. Agitate or shake all samples and controls gently (a 

roller shaker is recommended) for 2 hours. In 

order to increase the binding efficiency, the 

binding step may be extended overnight. 

7. Centrifuge the resin for 30 seconds at 

5,000–8,200 × g. Remove the supernatants with a 

narrow-end pipette tip. 

8. Wash the resin three times with 0.5 ml of TBS. 

Make sure all the supernatant is removed by using 

a Hamilton syringe or equivalent device. 

B. Elution of the FLAG-fusion protein 

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, pH 3.5. This is a fast and efficient elution 

method. Equilibration of the eluted protein with 

wash buffer may help preserve its activity. 

3. Elution with sample buffer for gel electrophoresis 

and immunoblotting. 

1. Elution with 3X 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 1 M NaCl at a 

concentration of 25 μg/μl. Dilute 5-fold with 

water to prepare a 3X FLAG stock solution 

containing 5 μg/μl of 3X FLAG peptide. For 

elution, add 3 μl of 5 μg/μl of 3X FLAG peptide 

stock solution to 100 μl 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 seconds 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 use, store the supernatants at 

2–8 °C. Store at –20 °C for long term storage. 

2. Elution with 0.1 M glycine HCl, pH 3.5 

The procedure should be performed at room 

temperature. Do not leave the resin in this buffer 

more than 20 minutes. 

a. Add 100 μl of 0.1 M glycine HCl, pH 3.5, buffer 

to each sample and control resin. 

b. Incubate the samples and controls with gentle 

shaking for 5 minutes at room temperature. 

c. Centrifuge the resin for 30 seconds at 

5,000–8,200 × g. Transfer the supernatants to 

fresh test tubes containing 10 μl of 0.5 M 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 use, 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 use. 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 50 kDa 

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, pH 6.8, with 2% 

SDS, 10% (v/v) glycerol, and 0.002% bromphenol 

blue) should be 5% or 50 mM, respectively. 

Note: SDS in the sample buffer will denature the M2 

antibody, and the ANTI-FLAG M2 affinity gel cannot be 

reused after treatment with the SDS-PAGE sample 

buffer. 

a. Add 20 μl of 2× 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 3 minutes. 

c. Centrifuge the samples and controls at 

5,000–8,200 × g for 30 seconds to pellet any 

undissolved agarose. 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 

against the fusion protein. 

5

6 

Reagent Compatibility Table 

Reagent Effect Comments 

Chaotropic agents 

(e.g., urea, 

guanidine HCl) 

Reducing agents 

(such as DTT, DTE, 

2-mercaptoethanol) 

TWEEN 20, 

5% or less 

TRITON X-100, 

5% or less 

IGEPAL CA-630, 

0.1% or less 

CHAPS, 

0.1% or less 

Digitonin, 

0.2% or less 

Sodium chloride, 

1.0 M or less 

Sodium dodecyl 

sulfate (SDS) 

0.1 M glycine HCl, 

pH 3.5 

Denatures the immobilized M2 

antibody 

Reduces the disulfide bridges 

holding the M2 antibody 

chains together 

Reduces nonspecific protein 

binding to the resin 










binding to the resin by 

reducing ionic interactions 

Denatures the immobilized M2 

antibody 

Elutes FLAG protein from the 

resin 

Deoxycholate Interferes with M2 binding to 

FLAG proteins 

Do not use any reagent that contains these types of 

components since it will denature the M2 antibody on the 

resin and destroy its ability to bind the FLAG fusion proteins. 

Low concentrations of urea (1 M or less) can be used. 

Do not use any reagent that contains these types of 

components since it will reduce the disulfide linkages in the 

M2 antibody on the resin and destroy its ability to bind the 

FLAG fusion proteins. 

May be used up to recommended concentration of 5%, but do 

not exceed. 

May be used up to recommended concentration of 5%, but do 

not exceed. 

May be used up to recommended concentration of 0.1%, but 

do not exceed. 





May be used up to recommended concentration of 1.0 M, but 


Do not use any reagent that contains this detergent in the 

loading and washing buffers since it will denature the M2 

antibody on the resin and destroy its ability to bind the FLAG 

fusion proteins. It is included in the sample buffer for removal 

of protein for immunoprecipitation, but the resin cannot be 

reused. 

Do not leave the column in glycine HCl for longer than 

20 minutes. Longer incubation times will begin to denature 

the M2 antibody 

Do not use any reagent that contains this detergent since it 

will inhibit the M2 antibody from binding to FLAG fusion 

proteins.

Troubleshooting Guide 

Problem Possible Cause Solution 

No signal is 

observed. 

Background is too 

high. 

FLAG fusion protein is not 

present in the sample. 

• Make sure the protein of interest contains the FLAG-tag 

by immunoblot or dot blot analyses. 

• Prepare fresh lysates. Avoid using frozen lysates. 

• Use appropriate protease inhibitors in the lysate or 

increase their concentrations to prevent degradation of 

the FLAG fusion protein. 

Washes are too stringent. • Reduce the number of washes. 

• Avoid adding high concentrations of NaCl to the mixture. 

• Use solutions that contain less or no detergent. 

Incubation times are 

• Increase the incubation times with the affinity resin (from 

inadequate. 

several hours to overnight). 

Interfering substance is • Lysates containing high concentrations of dithiothreitol 

present in sample. 

(DTT), 2-mercaptoethanol, or other reducing agents may 

destroy antibody function, and must be avoided. 

• Excessive detergent concentrations may interfere with 

the antibody-antigen interaction. Detergent levels in 

buffers may be reduced by dilution. 

Detection system is 

If Western blotting detection is used: 

inadequate. 

• Check primary and secondary antibodies using proper 

controls to confirm binding and reactivity. 

• Verify that the transfer was adequate by staining the 

membrane with Ponceau S. 

• Use fresh detection substrate or try a different detection 

system. 

Proteins bind nonspecifically to • Pre-clear lysate with Mouse IgG-Agarose (Catalog 

the ANTI-FLAG monoclonal Number A0919) to remove nonspecific binding proteins. 

antibody, the resin beads, or • After suspending beads for the final wash, transfer entire 

the microcentrifuge tubes. 

sample to a clean microcentrifuge tube before 

centrifugation. 

Washes are insufficient. • Increase the number of washes. 

• Prolong duration of the washes, incubating each wash for 

at least 15 minutes. 

• Increase the salt and/or detergent concentrations in the 

wash solutions. 

• Centrifuge at lower speed to avoid nonspecific trapping of 

denatured proteins from the lysate during the initial 

centrifugation of the affinity resin complexes. 

7

8 

References 

1. Brizzard, B.L., et al., BioTechniques, 16, 730 

(1994). 

2. Knappik, A., and Pluckthun, A., BioTechniques, 

17, 754 (1994). 

3. Chiang, C.M., and Roeder, R.G., Pept. Res., 6, 62 

(1993). 

4. Current Protocols in Molecular Biology, Ausubel 

F.M., et al. (John Wiley and Sons Inc., NY, 1998), 

pp. 10.15.1.-10.16.29 

5. Antibodies, A Laboratory Manual, Harlow, E. and 

Lane, D. (Cold Spring Harbor Laboratory Press, 

NY, 1988), pp. 514-517, 541-542, 547-549 

6. Reichelt, P., et al., Protein Expression and 

Purification, 46, 483–488 (2006). 

FLAG, ANTI-FLAG, and Trizma are registered 

trademarks of Sigma-Aldrich ® Biotechnology LP and 

Sigma-Aldrich Co. 

CelLytic and FLAG-BAP are trademarks of Sigma- 

Aldrich Biotechnology LP and Sigma-Aldrich Co. 

TRITON is a trademark of the Union Carbide Corp. 

TWEEN is a registered trademark of Uniqema, a 

business unit of ICI Americas, Inc. 

IGEPAL is a registered trademark of Rhone-Poulenc 

AG Co. 

Hamilton is a registered trademark of Hamilton Co. 

Coomassie is a registered trademark of Imperial 

Chemical Industries Ltd. 

Benzonase is a registered trademark of Merck KGaA, 

Darmstadt, Germany. 

BD,RM,DJ,CMH,KAT,MAM 08/10-1 

Sigma brand products are sold through Sigma-Aldrich, Inc. 

Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. 

Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. 

Please see reverse side of the invoice or packing slip.

ANTI-FLAG M2 Affinity Gel (A2220) - Technical ... - Sigma-Aldrich

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