49-1029

antibody from Invitrogen Antibodies

Targeting: MECP2 MRX16, MRX79, RTT

Western blot (Supportive data in Antibodypedia)

ELISA (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

49-1029

Provider

Invitrogen Antibodies

Product name

MECP2 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Other

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

50 µg

Concentration

Conc. Not Determined

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Whole cell extracts (40 μg) from HeLa cells transfected with MeCP2 siRNA (lane 2) and from an untransfected control (lane 1) were analysed by Western blot using the anti-MeCP2 antibody (Product # 49-1029) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Nuclear extracts (40 μg) from HeLa cells were analysed by Western blot using the anti-MeCP2 antibody (Product # 49-1029) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

ELISA

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Titer determination. To determine the titer, an ELISA was performed using anti-MeCP2 crude serum, purified anti-MeCP2 antibodies (Product # 49-1029), and the column flow through obtained from the antibody purification step. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be 1:15,000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the anti-MeCP2 antibody (Product # 49-1029) and the crude serum. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be: 1:32,900.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP assays were performed using human osteosarcoma (U-2 OS) cells, the anti-MeCP2 antibody (Product # 49-1029), and optimized PCR primer sets. Each ChIP assay used sheared chromatin from 1 million cells and 8.5 µg of anti-MeCP2 antibody (lanes 3 and 6), IgG (lanes 1 and 4), and beads only (lanes 2 and 5) were used as negative IP controls. The recovery, expressed as the percentage of input DNA, is shown here. A: Recovery of the RASSF1A promoter by the antibody against MeCP2, beads only or IgG B: Recovery of the CDC6 promoter (used as negative control) by the MeCP2 antibody, beads only or IgG.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP assays were performed using human osteosarcoma (U2OS) cells, the anti-MeCP2 antibody (Product # 49-1029) and optimized PCR primer sets. Sheared chromatin from 1x10^6 cells and 5 μg of antibody were used per ChIP experiment. IgG (1 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the ZMYND10 gene (used as a positive control) and CDC6 gene (used as a negative control). The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of MECP2 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of polyclonal MECP2 antibody (Product # 49-1029) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-rabbit coated Dynabeads (Product # 11204D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of MECP2 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of polyclonal MECP2 antibody (Product # 49-1029) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-rabbit coated Dynabeads (Product # 11204D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.