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Our Abpromise guarantee covers the use of ab2828 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/100 - 1/200.|
|IP||Use at an assay dependent concentration.|
|WB||Use at an assay dependent concentration. Detects a band of approximately 56 kDa (predicted molecular weight: 52.4 kDa).Can be blocked with Mouse MeCP2 peptide (ab4912). Recent lots of product ab2828 should detect the untruncated protein in human samples, 75 kDa band. Earlier lots detected the truncated version (55 kDa) in human and mouse samples. However in mouse samples, only a 55 kDa band is detected by all lots of the antibody. The difference between the earlier lots and the more recent lots is the antibodies were derived from different rabbits.|
|EMSA||Use at an assay dependent concentration. PubMed: 20385708|
|IHC-FoFr||Use at an assay dependent concentration.|
|IHC-P||1/200 - 1/2000.|
|ChIP||Use 5-25 µg for µg of chromatin. PubMed: 18653709|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 25409090|
Immunocytochemistry/Immunoflorescence of C2C12 cells labeling Methyl CpG Binding Protein 2 (green) with ab2828 at a dilution of 1/200. Cells with primary antibody (right) compared to negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature.Cells were probed with polyclonal antibody in 3% BSA-PBS and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Actin was stained using Alexa Fluor 554 (red) and nuclei were stained with Hoechst or DAPI (blue).
Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 5 µg of ab2828 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
Immunohistochemistry analysis of rat brain tissue staining MeCP2 with ab2828 (dilution 1/1000 in 3 % BSA-PBS). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab2828 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
ab2828 staining MeCP2 (green) in mouse hippocampus CA1 tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, permeablized with 0.1% Triton X-100 and blocked with 3% serum for 30 minutes at 23°C. The sample was incubated with primary antibody (1/200 in PBS + 3% BSA + 0.1% Triton X-100) at 4°C for 12 hours. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (H+L) polyclonal (1/1000) was used as the secondary antibody. Counterstained neuronal marker mouse anti-NeuN (red).
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