Anti-CBX1 / HP1 beta 抗体

• IP

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Immunoprecipitation - Anti-CBX1 / HP1 beta antibody (AB10478)

CBX1 / HP1 beta was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to CBX1 / HP1 beta and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab10478.
Secondary : Clean blot (HRP conjugate) at 1/1000 dilution.
Band : 20ka : CBX1 / HP1 beta.

All lanes:

Immunoprecipitation - Anti-CBX1 / HP1 beta antibody (ab10478)

Predicted band size: 21 kDa

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• WB

Lab

Western blot - Anti-CBX1 / HP1 beta antibody (AB10478)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : CBX1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : MCF7 whole cell lysate (20 μg)
Lane 4 : A431 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab10478 observed at 26 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab10478 was shown to recognize CBX1 when CBX1 knockout samples were used, along with additional cross-reactive bands. Wild-type and CBX1 knockout samples were subjected to SDS-PAGE. ab10478 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CBX1 / HP1 beta antibody (ab10478)

Predicted band size: 21 kDa

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• WB

Lab

Western blot - Anti-CBX1 / HP1 beta antibody (AB10478)

Lanes 1- 2 : Merged signal (red and green). Green - ab10478 observed at 26 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab10478 was shown to react with CBX1 / HP1 beta in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266356 (knockout cell lysate ab257383) was used. Wild-type HEK-293T and CBX1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab10478 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CBX1 / HP1 beta antibody (ab10478) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

CBX1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CBX1 (HP1 beta) knockout HEK-293T cell line (<a href='/products/cell-lines/human-cbx1-hp1-beta-knockout-hek-293t-cell-line-ab266356'>ab266356</a>)

Predicted band size: 21 kDa

Observed band size: 26 kDa

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• WB

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Western blot - Anti-CBX1 / HP1 beta antibody (AB10478)

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Western blot - Anti-CBX1 / HP1 beta antibody (ab10478) at 1/500 dilution

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Western blot - Recombinant Human CBX1 / HP1 beta protein (His tag N-Terminus) (<a href='/products/proteins-peptides/recombinant-human-cbx1-hp1-beta-protein-ab109847'>ab109847</a>) at 0.01 µg

Secondary

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Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 21 kDa

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Exposure time: 10s

• WB

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Western blot - Anti-CBX1 / HP1 beta antibody (AB10478)

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Western blot - Anti-CBX1 / HP1 beta antibody (ab10478) at 1 µg/mL

Lane 1:

HeLa whole cell lysate at 20 µg

Lane 2:

HeLa nuclear lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Lane 5:

HEK293 cell lysate at 20 µg

Lane 6:

HeLa whole cell lysate at 20 µg with CBX1 / HP1 beta peptide (<a href='/products/proteins-peptides/cbx1-hp1-beta-peptide-ab30861'>ab30861</a>)

Lane 7:

HeLa nuclear lysate at 20 µg with CBX1 / HP1 beta peptide (<a href='/products/proteins-peptides/cbx1-hp1-beta-peptide-ab30861'>ab30861</a>)

Lane 8:

A431 lysate at 20 µg with CBX1 / HP1 beta peptide (<a href='/products/proteins-peptides/cbx1-hp1-beta-peptide-ab30861'>ab30861</a>)

Lane 9:

MCF7 cell lysate at 20 µg with CBX1 / HP1 beta peptide (<a href='/products/proteins-peptides/cbx1-hp1-beta-peptide-ab30861'>ab30861</a>)

Lane 10:

HEK293 cell lysate at 20 µg with CBX1 / HP1 beta peptide (<a href='/products/proteins-peptides/cbx1-hp1-beta-peptide-ab30861'>ab30861</a>)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 21 kDa

Observed band size: 25 kDa,37 kDa,47 kDa

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• WB

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Western blot - Anti-CBX1 / HP1 beta antibody (AB10478)

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Western blot - Anti-CBX1 / HP1 beta antibody (ab10478) at 1 µg/mL

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PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

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Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 21 kDa

Observed band size: 27 kDa,90 kDa

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Exposure time: 4min

• WB

CiteAb

Western blot - Anti-CBX1 / HP1 beta antibody (AB10478)

CBX1 / HP1 beta western blot using anti-CBX1 / HP1 beta antibody ab10478. Publication image and figure legend from Weiss, T., Hergeth, S., et al., 2010, Epigenetics Chromatin, PubMed 20334638.

ab10478 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 ab10478 please see the product overview.

H1.2K187 methylation is cell cycle-independent and HP1 binds only to methylated H1.4K26. (a) H1.2K187me is cell cycle-independent. Quantitative MS analysis of H1 extracted from unsynchronized HEK293 cells and synchronized cells in G1 and M phase. A selected ion monitoring experiment was used on the ion 414.745 m/z (corresponding to H1K187 methylation) to monitor the levels of methylation. Normalisation was done relative to unsynchronized cells. (b) Recombinant HP1 binds to H1.4K26me2 but not to H1.2K187me2. Peptide affinity purification of recombinant HP1β and γ (GST fusion protein). The upper panels show the anti GST WB of the elution and lower panels the Coomassie staining of eluted fraction. (c) Endogenous HP1 binds to H1.4K26me2 but not H1.2K187me2. Western Blot analysis of peptide affinity purification of nuclear extracts (HeLa extracts) with HP1α/β specific antibody. (d) Glp1 does not bind to H1.4K26me2 or H1.2K187me2. Western Blot analysis of peptide affinity purification of nuclear extracts (HeLa extracts) with Glp1 specific antibody. (e) JMJD2D demethylates H1.4K26me2 only. Methylation assay with G9a and Glp1 after demethylation of peptides by JMJD2D (Rad).

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