Anti-RAB29 (phospho S72) antibody [EPR25173-80]

• WB

Lab

Western blot - Anti-RAB29 (phospho S72) antibody [EPR25173-80] (AB302702)

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

Rab29-S72 is phosphorylated by LRRK2 (PMID : 29125462).

Two bands detect in OE cell was due to endogenous expression Rab29 in HEK293 and overexpression Rab29 (PMID : 33135724).

All lanes:

Western blot - Anti-RAB29 (phospho S72) antibody [EPR25173-80] (ab302702) at 1/1000 dilution

Lane 1:

HEK-293T cells co-transfected with a human RAB29 expression vector containing a Myc-his tag and a human LRRK2 (Y1699C mutant) expression vector containing a flag tag, whole cell lysate at 20 µg

Lane 2:

HEK-293T cellls co-transfected with a human RAB29 (S72A mutant) expression vector containing a Myc-his tag and a human LRRK2 (Y1699C mutant) expression vector containing a flag tag, whole cell lysate at 20 µg

Lane 3:

HEK-293T cells transfected with a human RAB29 expression vector containing a Myc-his tag, 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: 20-30 kDa

false

Exposure time: 180s

• IP

Lab

Immunoprecipitation - Anti-RAB29 (phospho S72) antibody [EPR25173-80] (AB302702)

RAB29 (phospho S72) was immunoprecipitated from 0.35 mg HEK-293T cells co-transfected with a human RAB29 expression vector containing a Myc-his tag and a human LRRK2 (Y1699C mutant) expression vector containing a flag tag whole cell lysate with ab302702 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab302702 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-RAB29 (phospho S72) antibody [EPR25173-80] (ab302702) at 1/1000 dilution

Lane 1:

HEK-293T cells co-transfected with a human RAB29 expression vector containing a Myc-his tag and a human LRRK2(Y1699C mutant) expression vector containing a flag tag, whole cell lysate at 10 µg

Lane 2:

ab302702 at 1/30 IP in HEK-293T cells co-transfected with a human RAB29 expression vector containing a Myc-his tag and a human LRRK2(Y1699C mutant) expression vector containing a flag tag whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab302702 in HEK-293T cells co-transfected with a human RAB29 expression vector containing a Myc-his tag and a human LRRK2(Y1699C mutant) expression vector containing a flag tag whole cell lysate at 10 µg

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: 20-30 kDa

false

Exposure time: 67s

• Dot

Lab

Dot Blot - Anti-RAB29 (phospho S72) antibody [EPR25173-80] (AB302702)

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

Dot blot analysis of RAB29 (phospho S72) using ab302702 at 1/1000 followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

All lanes:

Dot Blot - Anti-RAB29 (phospho S72) antibody [EPR25173-80] (ab302702) at 1/1000 dilution

Lane 1:

RAB29 (phospho S72) peptide

Lane 2:

RAB29 non-phospho peptide

Lane 3:

RAB29 (phospho S72/T71) peptide

Lane 4:

RAB29 (phospho T71) peptide

Secondary

All lanes:

Dot 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 1/100000 dilution

false

Exposure time: 180s