All lanes : Anti-SIX1 + SIX6 + Six3 + SIX2 antibody [EPR23608-2] (ab277942) at 1/1000 dilution

Lane 1 : A-204 (human muscle rhabdomyosarcoma) whole cell lysate
Lane 2 : Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 32 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID:31438963).

Negative control: Caco-2.

Lysate loaded onto lane 1 was made freshly and used in WB immediately to minimize protein degradation.

Exposure time: 37 seconds.

 

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling SIX1+SIX2+SIX3+SIX6 with ab277942 at 1/200 dilution (2.57 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human skeletal muscle (PMID: 23840772). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized C2C12 (mouse myoblasts myoblast) cells labelling SIX1+SIX2+SIX3+SIX6 with ab277942 at 1/50 dilution (1µg, Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

SIX1+SIX2+SIX3+SIX6 was immunoprecipitated from 0.35 mg C2C12 (mouse myoblasts myoblast) whole cell lysate with ab277942 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab277942 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C2C12 whole cell lysate 10 µg

Lane 2: ab277942 IP in C2C12 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab277942 in C2C12 whole cell lysate

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

Exposure time: 24 seconds.

The bands beneath the target band is caused by degradation as demonstrated by WB data.

All lanes : Anti-SIX1 + SIX6 + Six3 + SIX2 antibody [EPR23608-2] (ab277942) at 1/1000 dilution

Lanes 1 & 3 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : C2C12 (mouse myoblasts myoblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 32 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

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

Lysate was made freshly and used in WB immediately to minimize protein degradation (lane 3). Lanes 1-2 are lysates that have experienced freeze-thaw cycles.

Exposure times: Lanes 1-2: 59 seconds; Lane 3: 26 seconds.

 

Immunohistochemical analysis of paraffin-embedded mouse skeletal muscle tissue labeling SIX1+SIX2+SIX3+SIX6 with ab277942 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse skeletal muscle (PMID: 23840772). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-SIX1 + SIX6 + Six3 + SIX2 antibody [EPR23608-2] (ab277942) at 1/1000 dilution

Lane 1 : Human skeletal muscle tissue lysate
Lane 2 : Mouse skeletal muscle tissue lysate
Lane 3 : Rat skeletal muscle tissue lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 5 : 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
Lane 6 : LADMAC (mouse bone marrow monocyte macrophage) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 32 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

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

Lysates used in this blot have experienced freeze-thaw cycles.

Exposure time: 3 minutes.

 

Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue labeling SIX1+SIX2+SIX3+SIX6 with ab277942 at 1/5000 dilution (0.103 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat skeletal muscle (PMID: 23840772). Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

All lanes : Anti-SIX1 + SIX6 + Six3 + SIX2 antibody [EPR23608-2] (ab277942) at 1/1000 dilution

Lane 1 : His-tagged human SIX1 recombinant protein, 20 ng
Lane 2 : E.coli extracts containing his-tagged human full length SIX2 recombinant protein at 15 µl
Lane 3 : E.coli extracts containing his-tagged human full length SIX6 recombinant protein at 20 µl
Lane 4 : Myc/DDK-tagged human fl length SIX3 HEK293T cell transient overexpression lysate at 20 µg
Lane 5 : Myc/DDK-tagged human fl length SIX4 recombinant protein 20 ng
Lane 6 : Myc/DDK-tagged human fl length SIX5 recombinant protein 20 ng

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 32 kDa

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

SIX1 recombinant protein was expressed from an E.coli expression system.

Exposure time: 15 seconds.