Flag-Catch-Aga™ Anti-DYKDDDDK (Flag) tag Alpaca nanobody Conjugated Agarose Beads

Overview

Description

Flag-Catch-Aga™ Anti-Flag tag nanobody (from alpaca VHH, Single Domain antibody) conjugated Agarose Beads

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

Product Picture 1

Figure 1. Alpaca antibodies are smaller than other mammalian antibodies. rabbit and mouse IgG consists a heavy (green) and light (blue) chain, with variable heavy (VH) and variable light (VL) domains. alpaca 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

Figure 2. Comparison of antibody types. Schematic representation of the comparison between (A) conventional antibodies, (B) alpaca heavy-chain antibodies, (C) and single domain antibodies.

Properties

Immunogen

This nanobody is produced from alpaca with a flag tagged protein.

Form

50% beads slurry

Agarose 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, it is from aplaca, 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

~ 90 µm

Binding Capacity

~1mg Flag tagged protein /ml beads suspension

Product Sheet

Applications

Application Image

Highlights

Protocols

Protocol for Immunoprecipitation (IP/Co-IP) of FLAG-Fusion Proteins from mammalian cell lysate (For soluble cytoplasmic protein)

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

 

Procedure

Attention:

1. If your target protein is soluble nuclear protein in nucleus (Excluding nuclear membrane integrated protein and DNA-binding protein), please use NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo, Cat no:78833) to extract nuclear protein. Or please use RIPA lysis buffer to lyse the cell, and add PIC, 1mM PMSF, 2.5mM Mgcl2 and 1mg/ml DNase I.

 

2. It is required to reserve 50 µl of protein extract for each sample as the INPUT control, which is directly used in the downstream WB experiment.

 

1. Harvest cells

1). For one immunoprecipitation reaction the use of ~106 - 107 mammalian cells (approx. one 10-cm dish) expressing a FLAG-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 mL ice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4°C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells. 

 

2. Lyse cells

1). Resuspend cell pellet in 500 µL ice-cold lysis buffer by pipetting.

Note: Add protease inhibitors and 1 mM PMSF into lysis buffer.

 

2). Place the tube on ice for 30 min with extensively pipetting every 10 min.

 

3). Centrifuge cell lysate at 14000x g for 10 min at +4°C. Transfer lysate(supernatant) to a pre-cooled tube. Discard pellet.

Note: At this point cell lysate may be put at -80°C for long-term storage.

 

3. Equilibrate beads

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

2). Pipette 25 µL bead slurry into 500 µL ice-cold cell extract. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.

 

4. Bind proteins

1). Add 25 µL equilibrated anti-FLAG agarose beads slurry (step 3) into 500 µL cell extract (step 2). Save 30 µL of cell extract for input of immunoblot analysis before IP. Incubation at a rotator for 1-3 hours at 4°C or overnight 4°C.

 

2). Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant.

 

5. Wash beads

1). Resuspend protein-antibody-beads complex in 1mL ice-cold wash buffer. Centrifuge at 2500x g for 5 min at +4°C. Discard supernatant and repeat wash at least three times.

 

6. Elute 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 50-100 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. Centrifugate the beads complex at 2500x g for 5 min at 4°C. 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 4 °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 to dissociate immunocomplex from anti-FLAG agarose beads. 

 

3. Anti-FLAG agarose beads immunocomplex can be centrifugated at 2500x g for 2 min at 4°C. 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. Centrifuge the resin for 30 seconds at 5,000–8,000g. 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.