All lanes : Anti-PMP22 antibody [EPR23112-110] (ab270400) at 1/1000 dilution

Lane 1 : Human leg nerve tissue lysate
Lane 2 : Human brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 18 kDa
Observed band size: 18,22 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

The expression profile observed is consistent with what has been described in the literature (PMID: 25429154; 7649472).
Negative control: Human brain (PMID: 7649472).

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

All lanes : Anti-PMP22 antibody [EPR23112-110] (ab270400) at 1/1000 dilution

Lane 1 : 293T (human embryonic kidney epithelial cell) transfected with an empty vector (vector control) containing a myc-His-tag®, whole cell lysate
Lane 2 : 293T transfected with PMP22 (WT) expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 18 kDa
Observed band size: 18,20 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

PMP22  was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) transfected with PMP22 (WT) expression vector containing a myc-His-tag®, whole cell lysate with ab270400 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270400 1/1000 dilution (0.58 μg/ml). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: 293T cells transfected with PMP22 (WT) expression vector containing a myc-His-tag®, whole cell lysate 10 μg

Lane 2: ab270400 IP in 293T transfected with PMP22 (WT) expression vector containing a myc-His-tag®, whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270400 in 293T transfected with PMP22 (WT) expression vector containing a myc-His-tag®, whole cell lysate. Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds

Immunohistochemical analysis of paraffin-embedded Human nerve tissue labeling PMP22 with ab270400 at 1/32000 dilution (0.018 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in Schwann cells of human nerve (PMID:7691737) is observed. The section was incubated with ab270400 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling PMP22  with ab270400 at 1/32000 dilution (0.018 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control: negative staining in human kidney (PMID:7649472).

The section was incubated with ab270400 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells transfected with a myc-tagged PMP22 expression construct. cells labelling PMP22 with ab270400 at 1/250 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged PMP22 expression construct is observed. 2233S Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 647 Conjugate) was used to counterstain tubulin at 1/100 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized 293T transfected with myc-his tagged PMP22 overexpression construct cells labelling PMP22 with ab270400 at 1/600 dilution (0.1 ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were surface stained with isotype control or ab270400. Then fixed with 2% PFA for 10min followed by intracellularly stained with anti-myc tag conjugated to Alexa Fluor® 647.