Tag-ChIP-IT®

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.

Prior to using the Tag-ChIP-IT^® Kit, the protein of interest is cloned into an expression vector containing the C-terminal AM-tag, such as Active Motif's pAM_1C Empty Vector (Catalog No. 53023). Following sequence verification of the cloned insert, cells are transfected for expression of the AM-tagged fusion protein. Once transfection and expression conditions have been optimized, chromatin is prepared from the transfected cells using the Tag-ChIP-IT Kit. Intact cells are fixed with a specially formulated formaldehyde buffer, which cross-links and preserves protein/DNA interactions. DNA is then sheared into small fragments using sonication and incubated with an antibody directed against the AM-tag. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following immunoprecipitation, the DNA cross-links are reversed, the proteins are removed by Proteinase K and the DNA is recovered and purified. ChIP enriched DNA can be used for either gene-specific or whole-genome analysis.

Tag-ChIP-IT^®

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.

Reproducible 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.

Estrogen Receptor (ER) cDNA was cloned into pAM_1C Empty Vector (Catalog No. 53023) and sequence verified. Transient transfections were performed into Ishikawa cells. Cells were induced with estradiol (E2) and chromatin was harvested according to the instructions in the Tag-ChIP-IT Kit. The AM-Tag antibody was used to immunoprecipitate the cross-linked AM-tag-ER fusion protein. Following reversal of cross-links, enriched DNA was submitted for next generation sequencing. Data was compared to published ChIP-Seq results using an anti-ER antibody in the same cell line and induction conditions. ChIP-Seq data shows the same ER peak profile with the AM-tag immunoprecipitation as endogenous ER. The binding sites detected with the AM-tag samples were further evaluated for binding motifs. Results show that the estrogen receptor motif was indentified in both Tag-ChIP-IT samples.

Detect Sequence Variants, Mutations and Truncations

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.

Full-length and truncated Androgen Receptor protein sequences were individually cloned into the pAM_1C empty vector. Separate transfections were performed and cells were either untreated or treated with R1881, a synthetic agonist of androgen receptor. Chromatin was prepared and immunoprecipitated with an antibody directed against the AM-Tag according to the instructions in the Tag-ChIP-IT Kit. Samples were then submitted for sequencing. Results show the KLK3 gene (also known as PSA, prostate-specific antigen). Peaks are observed with both the full-length and truncated androgen receptor in the presence of R1881, but no peaks are observed in the absence of the agonist.

Achieve Greater Sensitivity with the Highly Specific AM-Tag

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.

ChIP-Seq was performed with the AM-Tag antibody on chromatin prepared from Human samples. The sequencing results show an absence of DNA binding peaks which indicates there is little to no cross-reactivity of the AM-tag with human chromatin, thereby eliminating background signal in the Tag-ChIP-IT assay.

Contents & Storage

Tag-ChIP-IT

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

pAM_1C_JunD

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