Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

HeLA cells were stained with ab10811 in panel one. In panel two they were stained with ab10811 (green) DAPI (blue) and SH-CREST (red) which stains the centromeres. Fix 30 minutes on ice in 4% formaldehyde in PEM.Quench autofluorescence 2 x 5 minutes with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM.Permeablize 30 minutes with 0.5% TX-100 in PEM.Block 30 minutes in 5% milk in TBST.Primary antibody incubated overnight at 4oC diluted 1/100 in 5% milk in TBST.Secondary antibody incubated 1 hour at RT diluted in 5% milk in TBST.Post-fix 20 minutes on ice in 4% formaldehyde in PEM.Quench autofluorescence 2 x 5 minutes with ammonium chloride in PEM.Counterstain with DAPI in TBST.Mount with ProLong Gold antifade reagent from Invitrogen.Notes : Ample washing between each step. TBST = Tris buffered saline + 0.1% Tween. PEM = 80 mM K-PIPES pH 6.8 5 mM EGTA 2 mM MgCl2.Please note this image was from a different batch produced using hybridoma method

This image is courtesy of Scott Slattery and Mike Mancini

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized CBX1 KO HAP1(human chronic myelogenous leukemia cell)/Wild-type HAP1 cells labelling CBX1 / HP1 beta with ab10811 at 1/100 dilution followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution(Green).

Confocal image showing nuclear staining in parental HAP1 cell line (shown in green) and negative staining in CBX1 KO HAP1 cell line. The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems TCS SP8).

ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (Red).

Secondary antibody only control : Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded (A) Parental HAP1 (human chronic myelogenous leukemia near-haploid cell) pellet/(B) CBX1 knockout HAP1 cell pellet labeling CBX1 / HP1 beta with ab10811 at 1/1000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Nuclear staining on parental HAP1(human chronic myelogenous leukemia near-haploid cell) (image A) no staining on CBX1 KO HAP1 cell line (image B)
The section was incubated with ab10811 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human skin tissue labeling CBX1 / HP1 beta with ab10811 at 1/1000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Nuclear staining on human skin.
The section was incubated with ab10811 for 30 mins at room temperature followed by anti-rat IgG antibody (ab102248) for 8 mins during the LeicaDS9800 kit staining procedure.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling CBX1 / HP1 beta with ab10811 at 1/1000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Nuclear staining on human testis.
The section was incubated with ab10811 for 30 mins at room temperature followed by anti-rat IgG antibody (ab102248) for 8 mins during the LeicaDS9800 kit staining procedure.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• IP

Lab

Immunoprecipitation - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

CBX1 / HP1 beta was immunoprecipitated from 0.4 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab10811 at 1/50 dilution (2µg in 0.4mg lysates). Western blot was performed on the immunoprecipitate using ab10811 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CBX1 / HP1 beta antibody [MAC353] (<a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a>) at 1/50 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate (Input) at 10 µg

Lane 2:

HepG2 whole cell lysate

Lane 3:

Rat IgG2b isotype control (<a href='/en-us/products/primary-antibodies/rat-igg2b-kappa-monoclonal-rtk4530-isotype-control-ab18541'>ab18541</a>) instead of <a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a> in HepG2 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 21 kDa

false

Exposure time: 1s

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunofluorescence analysis of mouse erythroleukemia cell nuclei staining CBX1 / HP1 beta in heterochromatin with ab10811.

Cells were fixed with paraformaldehyde permeabilized using Triton X-100 and blocked for 30 min with 2.5% BSA. Cells were incubated with primary antibody (1/50) before incubating with a Cy3-conjugated goat anti-rat IgG to detect staining.Please note this image was from a different batch produced using hybridoma method

Image from Talasz H et al., J Biol Chem. 2005 Nov 18;280(46):38814-22. Epub 2005 Sep 15. Fig 1.; doi: 10.1074/jbc.M505563200; November 18, 2005 The Journal of Biological Chemistry, 280, 38814-38822.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling CBX1 / HP1 beta with ab10811 at 1/5000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Nuclear staining on rat testis.
The section was incubated with ab10811 for 30 mins at room temperature followed by anti-rat IgG antibody (ab102248) for 8 mins during the LeicaDS9800 kit staining procedure.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling CBX1 / HP1 beta with ab10811 at 1/5000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Nuclear staining on mouse testis.
The section was incubated with ab10811 for 30 mins at room temperature followed by anti-rat IgG antibody (ab102248) for 8 mins during the LeicaDS9800 kit staining procedure.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling CBX1 / HP1 beta with ab10811 at 1/100 dilution followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution(Green).

Confocal image showing nuclear staining in NIH/3T3 cell line (shown in green). The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems TCS SP8).

ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (Red).

Secondary antibody only control : Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

CBX1 / HP1 beta was immunoprecipitated from 0.4 mg ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) whole cell lysate with ab10811 at 1/50 dilution (2µg in 0.4mg lysates). Western blot was performed on the immunoprecipitate using ab10811 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CBX1 / HP1 beta antibody [MAC353] (<a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a>) at 1/50 dilution

Lane 1:

ES-D3 [D3]  (mouse blastocyst-derived embryonic stem cell) whole cell lysate (Input) at 10 µg

Lane 2:

ES-D3 [D3]  (mouse blastocyst-derived embryonic stem cell) whole cell lysate (+)

Lane 3:

Rat IgG2b isotype control (<a href='/en-us/products/primary-antibodies/rat-igg2b-kappa-monoclonal-rtk4530-isotype-control-ab18541'>ab18541</a>) instead of <a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a> in ES-D3 [D3] whole cell lysate (-)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 21 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

ab181602 was used as a GAPDH loading control at a 1/200000 dilution.

In Western blot anti-CBX1 antibody ab10811 was shown to bind specifically to CBX1. Target of interest was observed at 21kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in CBX1 knockout cell line (lane 2).

All lanes:

Western blot - Anti-CBX1 / HP1 beta antibody [MAC353] (<a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

CBX1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 21 kDa

false

Exposure time: 81s

• WB

Lab

Western blot - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-CBX1 / HP1 beta antibody [MAC353] (<a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 21 kDa

false

Exposure time: 125s

• WB

Lab

Western blot - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The identity of the higher MW bands are unknown.

All lanes:

Western blot - Anti-CBX1 / HP1 beta antibody [MAC353] (<a href='/en-us/products/primary-antibodies/cbx1-hp1-beta-antibody-mac353-ab10811'>ab10811</a>) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 21 kDa

false

Exposure time: 26s

• I-ELISA

Lab

Indirect ELISA - Anti-CBX1 / HP1 beta antibody [MAC353] - BSA and Azide free (AB324761)

This data was developed using ab10811, the same antibody clone in a different buffer formulation.

Indirect ELISA analysis of ab10811 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rat IgG (H+L) at 1/1000 dilution.

Antigen : Human CBX1 / HP1 beta.

Antigen concentration : 1000 ng/ml