ChIP Anti-bodies H3K4me2

In ChIP, the most vital step is the binding of an antibody and choosing the right antibody. The binding affinity of different types of immunoglobulins to protein A or G differs significantly. Henceforth, it is recommended to choose either protein A or protein G coated beads. If you do not see any product in the positive control, add 5–10 μg of chromatin and 1–5 μg of antibody to each IP reaction and incubate with antibody overnight and an additional 2 hr after adding Protein G/A beads. If no product in the experimental, add more DNA to the PCR reaction or increase the number of amplification cycles. Choose an alternate, ChIP-validated antibody if the antibody does not work.

3 Matching solutions
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 3 matching solutions for this experiment

Anti-dimethyl-Histone H3 (Lys4) Antibody

Merck Millipore

Upstream tips	Protocol tips	Downstream tips
	Infected RBCs were crosslinked with 1 % formaldehyde (Catalog number—28908, THERMO Scientific) for 10 min, lysed and sonicated in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 % SDS, 4 % NP-40, 1 mM PMSF) to obtain an average chromatin size of 200–400 bp. Chromatin was pre-cleared using 50 µl of a 50 % protein A Sepharose (GE healthcare) slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h.

Protocol tips
Infected RBCs were crosslinked with 1 % formaldehyde (Catalog number—28908, THERMO Scientific) for 10 min, lysed and sonicated in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 % SDS, 4 % NP-40, 1 mM PMSF) to obtain an average chromatin size of 200–400 bp. Chromatin was pre-cleared using 50 µl of a 50 % protein A Sepharose (GE healthcare) slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h.

Protocol tips

Infected RBCs were crosslinked with 1 % formaldehyde (Catalog number—28908, THERMO Scientific) for 10 min, lysed and sonicated in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 % SDS, 4 % NP-40, 1 mM PMSF) to obtain an average chromatin size of 200–400 bp. Chromatin was pre-cleared using 50 µl of a 50 % protein A Sepharose (GE healthcare) slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of IP buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X 100). Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab7766)

Abcam

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line. -Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of the presence of SDS.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line. -Do not place ethidium bromide in the agarose gel or the electrophoresis buffer, because of the presence of SDS.

Manufacturer protocol Publication protocol 1 Relevant paper

Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.	-It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.	-Once in solution, store 1M DTT at -20°C.

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment