Anti-RAB12 (phospho S106) antibody [MJF-D29764-507]

• WB

Collaborator

Western blot - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] (AB325602)

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

Lanes 1 - 2:

Western blot - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] (ab325602) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/rab12-phospho-s106-antibody-mjf-d29764-507-bsa-and-azide-free-ab325632'>ab325632</a>)

Lane 1:

Wild-type MEF (mouse embryo fibroblast) cells transfected with a mouse LRRK2 expression vector, whole cell lysate at 20 µg

Lane 2:

RAB12 knockout MEF cells transfected with a mouse LRRK2 expression vector, whole cell lysate 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: 27 kDa

false

Exposure time: 180s

• IP

Lab

Immunoprecipitation - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] (AB325602)

RAB12 (phospho S106) was immunoprecipitated from 0.35 mg 293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate with ab325602 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325602 at 1/325602 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate
Lane 2 : ab325602 at 1/30 IP in 293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325602 in 293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] (ab325602) at 1/30 dilution

Lane 1:

293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate at 10 µg

Lane 2:

ab325602 at 1/30 IP in 293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325602 in 293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 27 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] (AB325602)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa);
Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(20-300KDa);
Anti-RAB12 antibody (ab316770) (1 : 1000) (27KDa).

All lanes:

Western blot - Anti-RAB12 (phospho S106) antibody [MJF-D29764-507] (ab325602) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human wildtype RAB12 expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human LRRK2 (G2019S mutant) expression vector containing a his-tag and a human RAB12 (S106A mutant) expression vector containing a his-tag were treated with 100nM Calyculin A for 30min, whole cell lysate 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: 27 kDa,36 kDa,20-300 kDa

false

Exposure time: 1s