perform ChIP without protein-specific antibodies
Active Motif's AM-tag offers several advantages over other tags, such as FLAG, GFP and HA. AM-tag is specifically designed to give low background signal in ChIP experiments. Also, in contrast to larger GFP and Halo® tags, the low molecular weight and unstructured design of the AM-tag reduces the possibility of impeding on the DNA binding domain. In addition, we have designed a high specificity antibody to the tag to increase the pull down efficiency during immunoprecipitation.
Use the pAM_1C Empty Vector to clone your protein of interest, or add the AM-tag sequence to the expression vector of choice. Following transfection and expression of your tagged protein, the Tag-ChIP-IT Kit can be used to isolate chromatin and perform immunoprecipitations using an antibody directed against the AM-tag. The Tag-ChIP-IT method yields highly reproducible results with proven performance in both qPCR and ChIP-Seq analysis.
- Ideal for targets without ChIP antibodies or with low affinity antibodies
- Enables studies of isoforms, mutations and truncations
- AM-tag design maximizes enrichment efficiency of low abundance proteins
- AM-tag has little to no cross-reactivity with mammalian systems for reduced background
To learn more about Tag-ChIP-IT®, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.
|pAM_1C Empty Vector||20 µg||53023||¥3,190||Buy|
|pAM_1C_JunD Vector||50 µg||53044||¥3,190||Buy|
|pAM Forward Primer||250 pmol||53049||¥1,240||Buy|
|pAM Reverse Primer||250 pmol||53050||¥1,240||Buy|
|High Sensitivity Chromatin Preparation||16 rxns||53046||¥3,640||Buy|
|AbFlex® AM-Tag antibody (rAb)||100 µg||91111||¥4,240||Buy|
|FuGENE® HD Transfection Reagent||0.2 ml||32042||¥1,630||Buy|
How does the Tag-ChIP-IT® Kit work?
Simply use the pAM_1C Empty Vector to clone your protein of interest in-frame with the C-terminal AM-tag. Alternatively, the AM-tag sequence can be cloned into your expression vector of choice. Following transfection and expression of your tagged protein, the Tag-ChIP-IT Kit can be used to isolate chromatin and perform immunoprecipitations using AM-Tag antibody specific for the AM-tag.
Figure 1: Flow Chart of the Tag-ChIP-IT Assay.
Transcription factor ChIP is often challenging due to a lack of available antibodies that are capable of recognizing target-bound protein of interest post-fixation, or the inability of available antibodies to distinguish between protein isoforms. Tag-ChIP-IT® Kit overcomes these challenges by enabling ChIP without the use of target-specific antibodies. With Tag-ChIP-IT, the protein of interest is cloned in-frame with a C-terminal AM-tag sequence. Following transfection and expression of the tagged protein, chromatin can be isolated and immunoprecipitated using an antibody directed against the AM-tag. The unique AM-tag is specifically designed to minimize cross-reactivity with mammalian samples for reduced background signal. Additionally, the design maximizes exposure of the AM-tag during the immunoprecipitation reaction to increase the enrichment efficiency of low abundance transcription factors for more reliable and consistent ChIP results.
A comparison of the Tag-ChIP-IT method versus ChIP using an antibody against the endogenous protein of interest reveals the same ChIP-Seq profile between the two methods. Replicate experiments also confirm the reproducibility of Tag-ChIP-IT.
Figure 1: Comparison of endogenous and AM-tag ChIP-seq data for estrogen receptor binding motifs.
One of the challenges of studying the effects of sequence variants, mutations and truncations on gene regulation involves the lack of available antibodies capable of distinguishing between protein isoforms. With Tag-ChIP-IT, each protein variant can be cloned and tagged separately to enable individual analysis.
Figure 2: Tag-ChIP-IT can be used to distinguish between full-length and truncated Androgen Receptor.
Active Motif's unique AM-tag was specifically designed for use in ChIP. The AM-tag has minimal cross-reactivity with mammalian samples, thereby reducing background signal. Another benefit of the AM-tag is that the sequence is unstructured, which allows the tag to protrude from the protein of interest for maximum exposure during the immunoprecipitation. This increases the enrichment efficiency by allowing even low abundance transcription factors to be detected.
Figure 3: Specificity of the AM-Tag.
Contents & Storage
The Tag-ChIP-IT Kit contains the same optimized reagents as our ChIP-IT High Sensitivity® Kit to enable specific detection of low abundance proteins. The kit includes buffers, protease inhibitors, Protein G agarose beads, DNA purification columns and the AM-Tag antibody. For cloning, the pAM_1C Empty Vector is available separately. The pAM_1C_JunD Vector is also sold separately for use as an experimental control.
Please note that the Tag-ChIP-IT Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Agarose Beads after you have received this kit. This kit includes the following components:
- AM-Tag Antibody (1 µg/µl); Store at -20°C
- RNase A (10 µg/µl); Store at -20°C
- Proteinase K (10 µg/µl); Store at -20°C
- Blocker; Store at -20°C
- Carrier; Store at -20°C
- 10X PBS; Store at -20°C
- 100 mM PMSF; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Precipitation Buffer; Store at -20°C
- Fixation Buffer; Store at 4°C
- Protein G Agarose beads; Store at 4°C
- TE, pH 8.0; Store at RT
- Detergent; Store at RT
- 5 M NaCl; Store at RT
- Stop Solution; Store at RT
- Chromatin Prep Buffer; Store at RT
- ChIP Filtration Columns; Store at RT
- ChIP Buffer; Store at RT
- Wash Buffer AM1; Store at RT
- Elution Buffer AM4; Store at RT
- DNA Purification Binding Buffer; Store at RT
- 3 M Sodium Acetate; Store at RT
- DNA Purification Wash Buffer; Store at RT
- DNA Purification Elution Buffer; Store at RT
- DNA Purification Columns; Store at RT
pAM_1C Empty Vector
Please note that the pAM_1C Empty Vector is shipped lyophilized at room temperature. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.
- 20 µg pAM_1C Empty Vector; Store at -20°C
- 250 pmol pAM Forward primer; Store at -20°C
- 250 pmol pAM Reverse primer; Store at -20°C
Please note that the pAM_1C_JunD Vector is shipped lyophilized at room temperature. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.
- 50 µg pAM_1C_JunD Vector; Store at -20°C