Anti-p53 (mono methyl K372) antibody (ab16033)

Immunoprecipitation - p53 (mono methyl K372) antibody (ab16033)

Whole cell extracts derived from HEK293 cells stably expressing either Set7/9 wt or Set7/9 DN mutant were subjected to immunoprecipitation with anti-p53 monomethyl K372 antibody, ab16033.

The input and immunoprecipitated materials were resolved by SDS-PAGE and analyzed by immunoblotting using p53-specific antibody (Ab6-Oncogene).

Lanes 1 and 3 represent 2% of input materials for Set7/9wt and Set7/9DN mutant cell lines, respectively.
Lanes 2 and 4 represent immunoprecipitated materials from Set7/9wt and Set7/9DN extracts, respectively.

The data shows that p53 monomethyl K372 was succssfully IPed from the Set7/9 wt but not the Set 7/9 DN mutant cells.

NB:

The antibody was exhausted twice with 5 ug of control p53 peptide each time to eliminate non-specific binding to unmethylated p53. For westerns 5 ug of the blocking peptide was used per 10 ml of p53-K372me1 antibody in 1:5000 dilution.

Western blots were blocked in PBS with 2% non-fat milk for 2 hrs following the overnight incubation with p53-K372 me1-specific antibody (1:5000 and 5 ug of the blocking peptide). Secondary goat anti-rabbit antibody conjugated to peroxidase (BioRad) was used followed by incubation with ECL to develop the signal.

T. Ivanova, Tufts University

Immunoprecipitation - p53 (mono methyl K372) antibody (ab16033)

In vitro methylated or mock-methylated GST-p53 fragments (fragment = AAs300-393) were subjected to immunoprecipitation with anti-p53 monomethyl K372 antibody, ab16033.

(A) Western blotting with GST-specific antibody.
Lanes 1 and 2 – input materials representing 20% of methylated and mock-methylated GST-p53, respectively.
Lanes 3 and 4 – immunoprecipitated methylated and mock-methylated p53 proteins.

ab16033 was able to IP methylated but not unmethylated (mock methylated) GST-p53 fragments.

(B) Western blotting with anti-p53 monomethyl K372 antibody, ab16033.
Lanes 1 and 2 – input materials representing 20% of methylated and mock-methylated GST-p53, respectively.
Lanes 3 and 4 – immunoprecipitated methylated and mock-methylated p53 proteins.

ab16033 was able to IP and recognise by Western Blot methylated but not unmethylated (mock methylated) GST-p53 fragments.

NB:

The antibody was exhausted twice with 5 ug of control p53 peptide each time to eliminate non-specific binding to unmethylated p53. For westerns 5 ug of the blocking peptide was used per 10 ml of p53-K372me1 antibody in 1:5000 dilution.

Western blots were blocked in PBS with 2% non-fat milk for 2 hrs following the overnight incubation with p53-K372 me1-specific antibody (1:5000 and 5 ug of the blocking peptide). Secondary goat anti-rabbit antibody conjugated to peroxidase (BioRad) was used followed by incubation with ECL to develop the signal.

T. Ivanova, Tufts University

Western blot - p53 (mono methyl K372) antibody (ab16033)

All lanes : Anti-p53 (mono methyl K372) antibody (ab16033) at 1 µg/ml

Lane 1 : HEK293 Whole Cell Lysate Overexpressing p53
Lane 2 : HEK293 Whole Cell Lysate Overexpressing p53 with p53 peptide - mono methyl K372 (ab16203) at 1 µg/ml
Lane 3 : HEK293 Whole Cell Lysate Overexpressing p53 with p53 peptide - unmodified K372 (ab16204) at 1 µg/ml

Lysates/proteins at 5 µg per lane.

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

Performed under reducing conditions.

Predicted band size : 53 kDa
Observed band size : 50 kDa (why is the actual band size different from the predicted?)