Anti-CGBP antibody [EPR19199] - ChIP Grade

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed 293T (Human epithelial cell line from embryonic kidney) cells labeling CGBPwith ab198977 at 1/150 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling CGBP with ab198977 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin-Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab198977 at 1/250 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• ChIP

Unknown

ChIP - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab198977 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• IP

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Immunoprecipitation - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

CGBP was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab198977 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab198977 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate, 10μg (Input).

Lane 2 : ab198977 IP in HeLa whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A] -Isotype Control (ab172730) instead of ab198977 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977)

Predicted band size: 76 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Immunohistochemical analysis of paraffin-embedded Rat cerebellum tissue labeling CGBP with ab198977 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on rat cerebellum tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 cells labeling CGBPwith ab198977 at 1/150 dilution (red) compared with a Rabbit IgG,monoclonal-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at a dilution of 1/500 was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling CGBP with ab198977 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on mouse cerebrum tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling CGBP with ab198977 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected Anti-alpha Tubulin antibody - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab198977 at 1/250 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• WB

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Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977) at 1/2000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

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Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2/3 : 30 seconds; Lane 4 : 3 minutes; Lane 5 : 10 seconds.

All lanes:

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977) at 1/20000 dilution

Lane 1:

293T (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 10 µg

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Lane 4:

MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate at 10 µg

Lane 5:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

• WB

Supplier Data

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Blocking buffer : 5% NFDM/TBST.

Dilution buffer : 1% BSA/TBST.

This data is from our collaborator Hengyu-Fan's lab (Life Sciences Institute Zhejiang University).

All lanes:

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977) at 1/500 dilution

Lane 1:

WT HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lane 2:

CFP1(CGBP) KO HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L), HRP at 1/5000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 10 seconds; Lane 3/4/6 : 8 seconds; Lane 5 : 3 minutes.

Lanes 1 - 4:

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977) at 1/2000 dilution

Lanes 5 - 6:

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977) at 1/10000 dilution

Lane 1:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

L-929 (Mouse connective tissue fibroblast cell line) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 5:

L6 (Rat skeletal muscle cell line) whole cell lysate at 10 µg

Lane 6:

F9 (Mouse embryonic testicular cancer cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

• WB

Supplier Data

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 10 seconds; Lane 2/3/4 : 3 minutes.

All lanes:

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (ab198977) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

• WB

CiteAb

Western blot - Anti-CGBP antibody [EPR19199] - ChIP Grade (AB198977)

CGBP western blot using anti-CGBP antibody [EPR19199] - ChIP Grade ab198977. Publication image and figure legend from Tian, H., Billings, T., et al., 2018, PLoS Genet, PubMed 30365547.

ab198977 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab198977 please see the product overview.

Knocking out CXXC1 does not affect male fertility or testis histology.(A) CXXC1 is depleted in Stra8-cre CKO testes. Western blot of CXXC1 from adult B6, Cxxc1 het and CKO testicular extract. ß-tubulin was used as internal loading control. (B) CXXC1 is present in Sertoli cells but not in spermatocytes of Stra8-cre CKO mice. Immunostaining of CXXC1 on Cxxc1 het and CKO seminiferous tubule cross sections. Green, CXXC1; grey, DAPI. Long arrows, Sertoli cells; short arrows, spermatogonia; arrowhead, spermatocytes. Scale bar, 50 μm. (C) No change in fertility tests in CKO mice compared to B6 and Cxxc1 heterozygous controls. The number of viable pups for each genotype is shown. (D) Testis index (testis weight/body weight) is not changed in CKO mice compared to B6 and Cxxc1 heterozygous controls. (E) Normal histology of testis, epididymis and ovary is observed in both Cxxc1 control and CKO mice. Top panels, PAS staining of seminiferous tubule sections; scale bar, 100 μm. Middle panels, H&E staining of epididymis sections; scale bar, 200 μm. Bottom panels, H&E staining of 21 dpp ovary sections, Scale bar, 250 μm. Left panels, het control; right panels, Cxxc1 CKO with Stra8-Cre in male mice, and Ddx4-Cre in females. (F) No increased apoptosis is observed in testes of CKO mice. TUNEL staining in Cxxc1 het and CKO. Top panels, scale bar, 50 μm. Bottom panels, the apoptotic cell number is quantified as TUNEL positive cell number per seminiferous tubule. Data represent as mean ± SD, p = 0.94 by Student t-test.

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