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 : Anti-CBX1 / HP1 beta antibody (ab10478) at 1 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : HeLa nuclear lysate
Lane 3 : A431 cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : HEK293 cell lysate
Lane 6 : HeLa whole cell lysate with CBX1 / HP1 beta peptide (ab30861) at 1 µg/ml
Lane 7 : HeLa nuclear lysate with CBX1 / HP1 beta peptide (ab30861) at 1 µg/ml
Lane 8 : A431 lysate with CBX1 / HP1 beta peptide (ab30861) at 1 µg/ml
Lane 9 : MCF7 cell lysate with CBX1 / HP1 beta peptide (ab30861) at 1 µg/ml
Lane 10 : HEK293 cell lysate with CBX1 / HP1 beta peptide (ab30861) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size: 21 kDa
Observed band size: 25 kDa (why is the actual band size different from the predicted?)
Additional bands at: 37 kDa (possible cross reactivity), 47 kDa (possible cross reactivity)

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.

ab10478 at 1/200 staining human cervix cells by ICC/IF. The cells were fixed in 4% PFA for 15 minutes and permeabilized for 15 minutes in 0.5% Triton X100 in PBS, prior to incubation with the antibody fort 1 hour. A Cy2 ®  conjugated goat anti-rabbit antibody was used as the secondary.

See Abreview

Image courtesy of Human Protein Atlas

ab10478 staining CBX1 / HP1 in human testis, showing a distinct and strong staining pattern in ductus seminiferus cells. Paraffin embedded human skin tissue was incubated with ab10478 (1/400 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab10478 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

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Anti-CBX1 / HP1 beta antibody (ab10478) at 1 µg/ml + PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 27 kDa (why is the actual band size different from the predicted?)
Additional bands at: 90 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes