Anti-Bad (phospho S112) antibody [EPR1891(2)]

• IP

Lab

Immunoprecipitation - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

ab129192 (purified) at 1/30 immunoprecipitating Bad in HeLa cell lysate treated with Calcyculin A (Lane 1). For western blotting a HRP-conjugated anti-rabbit IgG was used as the secondary antibody (1/1000).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Bad (phospho S112) antibody [EPR1891(2)] (ab129192)

Predicted band size: 18 kDa

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• WB

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Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

All lanes:

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (ab129192) at 1/1000 dilution

Lane 1:

HeLa cell lysates, untreated at 10 µg

Lane 2:

HeLa cell lysates, treated with Calyculin A at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 18 kDa

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• WB

Lab

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) 1 : 1,000,000 dilution

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (ab129192) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate. (Untreated membrane) at 20 µg

Lane 2:

HeLa treated with 100 ng/ml Calyculin A for 30 min whole cell lysate. (Untreated membrane) at 20 µg

Lane 3:

HeLa treated with 100 ng/ml Calyculin A for 30 min whole cell lysate. (Phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 23 kDa

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Exposure time: 180s

• WB

Lab

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (ab129192) at 1/20000 dilution

Lane 1:

Untreated HeLa cell lysate at 10 µg

Lane 2:

HeLa cell lysate treated with Calcyculin A at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 18 kDa

Observed band size: 23 kDa

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• WB

Lab

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (ab129192) at 1/5000 dilution

Lane 1:

Untreated C6 cells at 10 µg

Lane 2:

C6 cells treated with Calcyculin A at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 18 kDa

Observed band size: 23 kDa

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• Dot

Lab

Dot Blot - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

Dot blot analysis of Bad (phospho S112) using ab129192 primary antibody at a dilution of 1/1000. ab97051 was used as a secondary antibody at a dilution of 1/100000.

Lane 1 : Bad (pS112) phospho peptide
Lane 2 : Bad non-phospho peptide

Blocking and diluting buffer : 5% NFDM/TBST

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Bad (phospho S112) antibody [EPR1891(2)] (AB129192)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.