Anti-GABARAPL1 + GABARAPL3 antibody [EPR18869] (ab191887) at 1/5000 dilution + Recombinant human GABARAPL1 full length protein at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 14 kDa
Observed band size: 43 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

In house recombinant Human GABARAPL1 is a GST-tagged full length protein (aa1-117).

All lanes : Anti-GABARAPL1 + GABARAPL3 antibody [EPR18869] (ab191887) at 1/5000 dilution

Lane 1 : Recombinant Human GABARAPL2 full length protein
Lane 2 : Recombinant Human GABARAPL3 full length protein
Lane 3 : Recombinant Human GABARAP (O95166) full length protein

Lysates/proteins at 0.01 µg per lane.

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

Predicted band size: 14 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

All three recombinant Human full length proteins contain aa1-117 with an N-terminal His-Tag® and were made in-house.

All lanes : Anti-GABARAPL1 + GABARAPL3 antibody [EPR18869] (ab191887) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal liver lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 14 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 15 seconds; Lane 2,3 and 4: 30 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 16650615).

All lanes : Anti-GABARAPL1 + GABARAPL3 antibody [EPR18869] (ab191887) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 14 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 5 seconds; Lane 2: 15 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 16650615).

All lanes : Anti-GABARAPL1 + GABARAPL3 antibody [EPR18869] (ab191887) at 1/1000 dilution

Lane 1 : Rat brain lysate
Lane 2 : Rat kidney lysate
Lane 3 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 5 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 14 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID: 16650615).

GABARAPL1 + GABARAPL3 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab191887 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab191887 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate 10ug (Input).

Lane 2: ab191887 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab191887 in HeLa whole cell lysate.

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

Exposure time: 30 seconds.