All lanes : Anti-beta Catenin non-phospho(active) S37/T41 antibody [EPR23969-131] (ab246504) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : CTNNB1 (beta Catenin) knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate
Lane 4 : CTNNB1 (beta Catenin) knockout HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBST

The molecular weight observed is consistent with what has been described in the literature (PMID: 16288032).

Lanes 1-4: Merged signal (red and green). Green - ab246504 observed at 92 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

Lanes 1-2: ab246504 Anti-beta Catenin (non-phospho(active) S37/T41) antibody [EPR23969-131] was shown to specifically react with beta Catenin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255352 (knockout cell lysate ab263756) was used.

Lanes 3-4: ab246504 Anti-beta Catenin (non-phospho(active) S37/T41) antibody [EPR23969-131] was shown to specifically react with beta Catenin in wild-type HAP1 cells. Loss of signal was observed when knockout sample was used.

Wild-type and beta Catenin knockout samples were subjected to SDS-PAGE. ab246504 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

 

An extra band around 45 kDa was observed.

 

All lanes : Anti-beta Catenin non-phospho(active) S37/T41 antibody [EPR23969-131] (ab246504) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 3 : LLC1 (mouse lung carcinoma ) whole cell lysate
Lane 4 : C6 (rat glial tumor glial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration:  5% NFDM/TBST

Exposure time 92 seconds

An extra band around 45 kDa was observed.

All lanes : Anti-beta Catenin non-phospho(active) S37/T41 antibody [EPR23969-131] (ab246504) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

An extra band around 45 kDa was observed.

 

 Exposure time: 3 minutes

 

Dot blot analysis of beta Catenin (non-phospho(active) S37/T41) using ab246504 at 1:1000 (0.541 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: beta Catenin non-phospho-Ser33/Ser37/Thr41 peptide (aa 29-44)
Lane 2: beta Catenin non-phospho-Ser33/Ser37/Thr41 peptide (aa 31-47)
Lane 3: beta Catenin phospho-Ser33 non-phospho-Ser37/Thr41 peptide (aa 29-44)
Lane 4: beta Catenin phospho-Ser37 non-phospho-Ser33/Thr41peptide (aa 29-44)
Lane 5: beta Catenin phospho-Thr41 non-phospho-Ser33/Ser37 peptide (aa 29-47)
Lane 6: beta Catenin phospho-Ser33/Ser37 non-phospho-Thr41 peptide (aa 29-47)
Lane 7: beta Catenin phospho-Ser33/Thr41 non-phospho-Ser37 peptide (aa 29-47)
Lane 8: beta Catenin phospho-Ser37/Thr41non-phospho-Ser33 peptide (aa 29-47)
Lane 9: beta Catenin phospho-Ser33/Ser37/Thr41peptide (aa 29-47)

Exposure time: 81 seconds

Blocking and diluting buffer and concentration: 5% NFDM/TBST

beta Catenin (non-phospho(active) S37/T41) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab246504 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab246504 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab246504 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab246504 in HeLa whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds