All lanes : Anti-CBX1 / HP1 beta antibody (ab10478) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CBX1 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

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.

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.

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

Anti-CBX1 / HP1 beta antibody (ab10478) at 1/500 dilution + Recombinant Human CBX1 / HP1 beta protein (ab109847) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 21 kDa


Exposure time: 10 seconds