Anti-Myc tag antibody [9E11]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Myc tag antibody [9E11] (AB56)

Overlay histogram showing HL60 cells stained with ab56 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse DyLight^® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IP

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Immunoprecipitation - Anti-Myc tag antibody [9E11] (AB56)

Myc tag was immunoprecipitated using 0.5mg CHO overexpressing Stra8 whole cell lysate, 5μg of Mouse monoclonal to Myc tag 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, CHO overexpressing Stra8 whole cell lysate 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°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab56.

Secondary : Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band : 49KDa; Myc tag

All lanes:

Immunoprecipitation - Anti-Myc tag antibody [9E11] (ab56)

Predicted band size: 49 kDa

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Exposure time: 20min

• WB

Lab

Western blot - Anti-Myc tag antibody [9E11] (AB56)

All lanes:

Western blot - Anti-Myc tag antibody [9E11] (ab56) at 1 µg/mL

Lane 1:

HEK293 cell lysate at 20 µg

Lane 2:

Western blot - Recombinant Human c-Myc protein (<a href='/en-us/products/proteins-peptides/recombinant-human-c-myc-protein-ab84132'>ab84132</a>) at 0.1 µg

Lane 3:

Western blot - E. coli Positive Control (Escherichia coli ) Whole Cell Lysate (<a href='/en-us/products/cell-lysates/e-coli-positive-control-escherichia-coli-whole-cell-lysate-ab5395'>ab5395</a>) at 0.1 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG H&L Pre-Adsorbed (HRP) at 1/5000 dilution

Predicted band size: 49 kDa

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• WB

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Western blot - Anti-Myc tag antibody [9E11] (AB56)

SDS-PAGE with Coomassie Blue staining (Left) and Western-blot (Right)

All lanes:

Western blot - Anti-Myc tag antibody [9E11] (ab56) at 1/500 dilution

All lanes:

Western blot - Recombinant Human c-Myc protein (<a href='/en-us/products/proteins-peptides/recombinant-human-c-myc-protein-ab84132'>ab84132</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 48 kDa

true

Exposure time: 4min

• WB

Unknown

Western blot - Anti-Myc tag antibody [9E11] (AB56)

All lanes:

Western blot - Anti-Myc tag antibody [9E11] (ab56) at 1/500 dilution

All lanes:

Western blot - Recombinant Human c-Myc protein (<a href='/en-us/products/proteins-peptides/recombinant-human-c-myc-protein-ab84132'>ab84132</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 49 kDa,68 kDa

Observed band size: 65 kDa

true

Exposure time: 4min