Lanes 1-2 : Coomassie stain
Lanes 3-4 : Anti-NF-kB p65 (acetyl K310) antibody (ab19870) at 1/500 dilution

Lanes 1 & 3 : Acetylated p65 protein
Lanes 2 & 4 : K310R mutant protein

Lysates/proteins at 2 µg per lane.

Secondary
Lanes 3-4 : IRDye® 800CW Goat anti-Rabbit IgG (H + L) at 1/15000 dilution

Predicted band size: 65 kDa

Observed band size: 75kDa

The p65 band runs higher in this blot because the protein contains a myc-tag.

All lanes : Anti-NF-kB p65 (acetyl K310) antibody (ab19870) at 1/1000 dilution

Lane 1 : HCT116 whole cell lysate treated with DMSO for 24 hrs (control)
Lane 2 : HCT116 whole cell lysate treated with 2 µM SAHA for 24 hrs
Lane 3 : MEF whole cell lysate treated with DMSO for 24 hrs (control)
Lane 4 : MEF whole cell lysate treated with 2 µM SAHA for 24 hrs

Lysates/proteins at 60 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 65 kDa
Observed band size: 65 kDa
Additional bands at: 140 kDa (possible non-specific binding), 15 kDa (possible non-specific binding), 40 kDa (possible non-specific binding), 45 kDa (possible non-specific binding), 90 kDa (possible non-specific binding)


Exposure time: 2 minutes

See Abreview

Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3h at room temperature. Exposure time: 75 min normal ECL. This Dot blot demonstrates that ab19870 recognized upto 10ng of purified peptide on a PVDF membrane.

Western Blot with ab19870 after p65 Immunoprecipitation: rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3 hours at room temperature. Exposure time: 1 min normal ECL. Tested samples: nuclear extracts (180 µg) of immortalized p65-/- mouse cells, complemented with the empty vector (pRRL), wild-type p65 (Wt) and non-acetylatable K310 (K310R). The samples tested were treated with deacetylase inhibitors HDACi (TSA + Nicotinamide) and TNF-alpha. The samples were immunoprecipitated with 2µg of alpha-p65 and subsequently analysed by Western blot with Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870). Predicted band size = 65kDa, Observed band size = 75kDa. The p65 band runs higher in this SDS-PAGE blot as it contains a myc-tag. 

All lanes : Anti-NF-kB p65 (acetyl K310) antibody (ab19870) at 2.5 µg/ml

Lane 1 : pRRL untreated
Lane 2 : pRRL HDACi
Lane 3 : pRRL HDACi + TNF
Lane 4 : Wt untreated
Lane 5 : Wt HDACi
Lane 6 : Wt HDACi + phorbol myristate acetate
Lane 7 : K310R untreated
Lane 8 : K310R HDACi
Lane 9 : K310R HDACi + phorbol myristate acetate

Lysates/proteins at 75 µg per lane.

Developed using the ECL technique.

Predicted band size: 65 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 1 hour

ab19870 recognizes Rabbit polyclonal to NF-kB p65 (acetyl K310) specifically at ~75kDa (indicated by the arrow) is this SDS-PAGE blot. The p65 band runs higher than 65kDa in this SDS-PAGE blot as it contains a myc-tag. We are sure that the band at ~75kDa is p65 since p65 specific antibodies detect the same band in IP and WB and there is no signal in the p65 knock-out cell line with ab19870. A number of additonal bands are recognized by ab19870 when tested with endogenous p65 from whole cell extracts, we do not know the identity of these bands.

Tested samples: nuclear extracts (75µg) of immortalized p65-/- mouse cells, complemented with the empty ve

Anti-NF-kB p65 (acetyl K310) antibody (ab19870) at 1 µg/ml + Lung (Rat) Tissue Lysate at 10 µg

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

Performed under reducing conditions.

Predicted band size: 65 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?
Additional bands at: 15 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes