PA5-27632

antibody from Invitrogen Antibodies

Targeting: POLR1G A34.5, ASE-1, CAST, CD3EAP, PAF49, RPA34

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (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

PA5-27632

Provider

Invitrogen Antibodies

Product name

PAF49 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant protein fragment

Description

Recommended positive controls: A549, H1299, HCT116.

Concentration

0.55 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PAF49 using Whole cell extracts (30 µg). Samples were loaded onto a 10% SDS-PAGE gel and probed with a PAF49 polyclonal antibody (Product # PA5-27632) at a dilution of 1:1000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of PAF49 was performed by separating 30 µg of various whole cell extracts by 10% SDS-PAGE. Proteins were transferred to a membrane and probed with a PAF49 Polyclonal Antibody (Product # PA5-27632) at a dilution of 1:1000.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of PAF49 was performed in A431 cells fixed in 4% paraformaldehyde at RT for 15 min. Green: PAF49 Polyclonal Antibody (Product # PA5-27632) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of PAF49 was performed in HeLa cells fixed in ice-cold MeOH for 5 min. Green: PAF49 Polyclonal Antibody (Product # PA5-27632) diluted at 1:1000. Red: alpha Tubulin, a cytoskeleton marker. Blue: Hoechst 33342 staining.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Paraffin) analysis of PAF49 was performed in paraffin-embedded human ovarian cancer tissue using PAF49 Polyclonal Antibody (Product # PA5-27632) at a dilution of 1:500.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin immunoprecipitation analysis of PAF49 was performed using cross-linked chromatin from 1x10^6 HCT116 colon carcinoma cells treated with serum for 0 and 30 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 1.0 µL/100 µL well volume of a PAF49 polyclonal antibody (Product # PA5-27632). Chromatin aliquots from ~1x10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify exon-1 of the RNA5-8S5 gene or -20 kb upstream of the LAMC1 gene. 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. A schematic representations of the RNA5-8S5 and LAMC1 loci are shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by RNA5-8S5 and LAMC1 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

RNA immunoprecipitation (RIP) western of CD3EAP 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 CD3EAP antibody (Product # PA5-27632) 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.