Flag-Catch-Mag™ Anti-DYKDDDDK (FLAG) tag Alpaca nanobody conjugated Magnetic Beads

Overview

Description

Flag-Catch-Mag™ Anti-Flag tag nanobody (amino acid sequence is derived from alpaca VHH, Single Domain antibody) conjugated Magnetic Beads

It is recombinantly expressed in yeast.

Advantage:

This is the only anti-flag alpaca nanobody conjugated beads in the world, not Fab conjugated beads from mice or rabbits. This anti-flag alpaca nanobody is better than Fab due to its higher stability and affinity. More importantly, downstream WB will not be interfered with antibody light and heavy chains.

 

On the contrary, mouse Fab will still be bound by downstream secondary antibodies, resulting in interference with light and heavy chains.

Reactivity

This product is useful tool for IP/CoIP/ChIP of Flag-tagged proteins, and it recognizes Flag-tags placed at N-terminal, C-terminal, or internal site of the fusion protein.

Tested applications

Immunoprecipitation (IP), 

Co-Immunoprecipitation (Co-IP), 

Chromatin Immunoprecipitation (ChIP) 

RNA Binding Protein Immunoprecipitation (RIP)

Enzyme assays

Mass spectrometry

Affinity purification

 

In IP, 25-50 µL of beads suspension for ~500-1000 µL of crude total cell lysate solution.

Optimal dilutions/concentrations should be determined by the end user.

Product Picture

Specificity

This antibody detects Flag-tagged proteins exogenously expressed in cells or E. coli. This antibody also detects 3*Flag-tagged proteins.

Product Picture 1

Figure 1. Alpaca antibodies are smaller than other mammalian antibodies. Mammalian IgG consists a heavy (green) and light (blue) chain, with variable heavy (VH) and variable light (VL) domains that facilitate the memory-like binding properties of antibodies. Camelid antibodies consist only a heavy chain (green) with a single variable heavy (VHH) domain for target recognition. This VHH domain can be expressed alone without the heavy chains, forming a single domain VHH.

 

Product Picture 2

Properties

Immunogen

DYKDDDDK synthetic peptide.

Form

Magnetic beads Conjugated anti-Flag nanobody, the product is supplied in 10 mM PBS, pH 7.4, and 0.02% (w/v) sodium azide as preservative.

Storage instruction

Stored at 4 °C. Do not freeze

Host

This antibody is nanobody, amino acid sequence is derived from alpaca VHH, it is recombinantly expressed in yeast.

Its advantages are as below:

 

• Fast, reliable & efficient one-step immunoprecipitation

 

• Ready-to-use, high stability, high affinity

 

• No heavy & light antibody chains interference in downstream WB

 

• Stable under harsh washing conditions

 

• Suitable for downstream mass spec, CoIP, ChIP, RIP, protien purification and so on

 

• Works in samples from: mammals, plants, bacteria, yeast, insects etc.

Structure Image

Beads Diameter

~ 40 µm

Binding Capacity

~1mg Flag tagged protein /ml beads suspension

Product Sheet

Applications

Application Image

Figure 1. A schematic representation of benefits (Alpaca Antibody Advantage)

 

1. No interfering of traditional antibody heavy and light chains in your downstream WB and mass spectrometry analysis
2. One step immunoprecipitation
3. Highly specific binding
4. Low background
5. High affinity
6. High stability
7. Easy elution of native proteins

Highlights

Figure 2. A schematic representation of benefits (no interfering of traditional antibody heavy and light chains)

Protocols

Protocol for Immunoprecipitation (IP/CoIP) of Flag-Fusion Proteins from Mammalian Cell Lysate

 

This protocol is intended for immunoprecipitation of flag tagged fusion proteins for analysis by western immunoblot or activity assay.

 

Solutions and Reagents

1X Cell Lysis Buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100. Recommend adding 1 mM PMSF before use.

 

1X Wash Buffer: TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.5)

 

5X SDS Sample Loading Buffer

 

1. Preparing Cell Lysates

1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS. Remove PBS and add 0.5 mL-1mL 1X ice-cold cell lysis buffer (added Protease Inhibitor Cocktail and PMSF) to each plate (10 cm,106-107 cells) and incubate the plates on ice for 30-40 minutes with rotation at 4°C.

2. Scrape lysed cells off the plates and transfer to microcentrifuge tubes. Keep on ice.

3. Sonicate samples on ice three times for 5 seconds each (optional step).

4. Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.

 

Attention: If the target protein is nuclear protein, please use RIPA lysis buffer to lyse the cell, and add PIC, 1mM PMSF, 2.5mM Mgcl2 and 1mg/ml DNase I.

 

2. Equilibrate Beads

1. Resuspend the beads by inverting the product tube or gently pipetting up and down. Do not Vortex the beads.

2. Pipette 25 µL bead slurry into an EP tube (1.5 mL),then add 1mL ice-cold wash buffer. Shake gently by your hand for 1-2 minutes. Do not vortex the beads.

3. Magnetic separation for 60 seconds. 

4. Discard supernatant and repeat wash three times.

 

3. Immunoprecipitation

1. Take 500 μL cell lysate and add 25 µL of the antibody conjugated magnetic beads suspension, incubate with rotation for 1-3 hours at 4°C or overnight at 4°C.

 

4. Wash the protein-nanobody-beads complex

NOTE: if Co-IP interacting proteins are researched, please reduce the number of washes, and lower the ionic strength of the wash buffer.

 

Add 1.0 mL of Wash Buffer and suspend the beads complex, magnetic separation, then discard the supernatant. Repeat above steps at least four times.

 

5. Elution for Downstream Analysis

Elution of the FLAG fusion proteins - Three elution methods are recommended according to protein characteristics or further usage: 

 

Option A: Native Elution

Elution under acidic conditions with 0.2 M glycine-HCl (pH 2.5). This is a fast and efficient elution method. Neutralization of the eluted proteins with neutralizing buffer (1 M Tris, pH 10.4) may help preserve its activity. Neutralizing buffer needs to be placed in the collection tube in advance.

 

Attention: It is necessary to make a preliminary experiment in advance to determine how much neutralization buffer is needed to neutralize glycine-HCl

 

Option B: Denaturing Elution for SDS-PAGE

Elution with sample loading buffer under denaturing conditions for gel electrophoresis and immunoblotting.

 

Option C: Protein elution under native conditions by competition with 3xFLAG peptide.
The elution efficiency is very high using this method.

 

Option A: Elution with 0.2 M Glycine-HCl (pH 2.5) - The procedure should be performed at room temperature. Note:Do not leave the beads in this buffer more than 20 minutes.

 

1. Add 50 µL-100 µL of 0.2 M Glycine-HCl (pH 2.5), to each sample and control beads complexes.

 

2. Incubate the samples and controls with constantly pipette up and down for 1-3 minutes at room temperature. (Note: Do not turn the tube upside down to prevent the complex from sticking to the tube wall)

 

3. Place tube in the appropriate magnetic separator to collect the beads. Transfer the supernatants to fresh tubes containing about 5 µL of neutralizing buffer. Then use a pH-indicator paper (pH 6.4 - 8.0) to make sure the pH is 7.4. Be careful not to transfer any beads.

 

4. Repeat steps 1 – 3 in order to improve elution efficiency, pooling eluates in same tube or collect eluate through different tubes. 

 

5. For immediate use, store the eluates at 2-8 °C. Store at –20 °C for long term storage. 

 

Option B: Elution with SDS-PAGE Sample Loading Buffer

1. Resuspend each sample with 30 µL 1X SDS sample loading buffer (6 µL 5X SDS sample loading buffer can be added into 24 µL cell lysis buffer). Vortex.

 

2. Boil the sample and control tubes for 10 minutes at 95 – 100°C. 

 

3. Place tubes in the magnetic separator to collect the beads. Transfer the supernatants to fresh tubes. The samples and controls are ready for loading on SDS-PAGE and immunoblotting using anti-flag or specific antibodies against the fusion protein.

 

Option C: Protein elution under native conditions by competition with 3xFLAG peptide. The elution efficiency is very high using this method.

 

1. Prepare 3X FLAG elution solution. 

Prepare 3X FLAG peptide stock solution (5 mg/mL). 

Dissolve 3X FLAG peptide (ENGIBODY, Cat. No: AT1968) in TBS (50mM Tris HCL, 150 mM NaCl, pH7.4) to a final concentration of 5 mg/mL. For extended storage after reconstitution, store at –20 °C in with 50% glycerol. Avoid repeated freeze-thaw.

 

Prepare 3X FLAG peptide working solution (1 mg/mL).

Dilute 5-fold with TBS to prepare a 3X FLAG peptide working solution containing 1 mg/mL of 3X FLAG peptide. 

 

2. Add 100 µL of 3X FLAG elution working solution to each sample and control beads.

 

3. Incubate the samples and controls with gentle shaking for 30-60 minutes at 2–8 °C.

 

4. Separation by magnetic rack. Transfer the supernatants to fresh test tubes. Be careful not to transfer any beads.

 

5. For immediate use, store the supernatants at 2–8 °C. Store at –20 °C for long term storage.