MA5-14107

antibody from Invitrogen Antibodies

Targeting: CDC25C CDC25, PPP1R60

Western blot (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-14107

Provider

Invitrogen Antibodies

Product name

Cdc25C Monoclonal Antibody (25C14 (TC-14))

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

MA5-14107 targets CDC25C in IF, IP, ChIP and WB applications and shows reactivity with Human samples.

Antibody clone number

25C14 (TC-14)

Concentration

0.2 mg/mL

References

Submitted references

HYAL1 hyaluronidase in prostate cancer: a tumor promoter and suppressor.

Lokeshwar VB, Cerwinka WH, Isoyama T, Lokeshwar BL
Cancer research 2005 Sep 1;65(17):7782-9

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of CDC25C was performed with 10 µg of HeLa cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against CDC25C (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. CDC25C was detected at ~ 55 kDa using CDC25C Mouse monoclonal antibody (Product # MA5-14107) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Mouse (Product # 31437) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to Cyclophilin B (21 kDa). CDC25C Antibody (Product # MA5-14107) confirms silencing of CDC25C expression.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry analysis of CDC25C (25C14 (TC-14)) showing staining in the nucleus and weak cytoplasm of paraffin-embedded human testis tissue (right) compared to a negative control without primary antibody (left). 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 a CDC25C Antibody (25C14 (TC-14)) Mouse Monoclonal Antibody (Product # MA5-14107) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°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.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin immunoprecipitation analysis of CDC25C was performed using cross-linked chromatin from 1x10^6 HCT116 colon carcinoma cells treated with serum for 0, 15, 30, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0ul/100ul well volume of a CDC25C monoclonal antibody (Product # MA5-14107). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1ul of eluted DNA in 2ul SYBR real-time PCR reactions containing primers to amplify exon 3-3 of human Bcl-x (hBcl-x) or exon 1 of human NR4A3 (hNR4A3). PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. Schematic representations of the Bcl-x and NR4A3 loci are shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions). Regions amplified by Bcl-x and NR4A3 primers are represented by black bars. Data courtesy of the Innovators Program.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of CDC25C using CDC25C Monoclonal Antibody (Product # MA5-14107) on denatured Human HeLa Cells.