All lanes : Anti-DMC1 antibody [EPR23207-130] (ab245217) at 1/1000 dilution

Lane 1 : Mouse testis lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat testis lysate
Lane 4 : Rat spleen lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 38 kDa
Observed band size: 38 kDa

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 22156371). 

Negative control: Spleen (PMID: 21085687). 

Exposure time: 26 seconds

All lanes : Anti-DMC1 antibody [EPR23207-130] (ab245217) at 1/1000 dilution

Lane 1 : His-tagged mouse DMC1 recombinant protein (aa2-340)
Lane 2 : His-tagged mouse Rad51 recombinant protein (aa2-339)

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

Predicted band size: 38 kDa
Observed band size: 38 kDa

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

No cross-reactivity with mouse Rad51 is observed. 

Exposure time: Lane 3 seconds; Lane 2:81 seconds.

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Rat testis tissue labeling VSIG4 with ab245217 at 1/100 (6.7 µg/ml) dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution.

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Mouse testis tissue labeling VSIG4 with ab245217 at 1/100 (6.7 µg/ml) dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution.

DMC1 was immunoprecipitated from 0.35 mg Mouse testis lysate 10ug with ab245217 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab245217 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: Mouse testis lysate 10ug

Lane 2: ab245217 IP in Mouse testis lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab245217 in Mouse testis lysate

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

Exposure time: 30 seconds

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling DMC1 with ab245217 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on mouse cerebrum. The section was incubated with ab245217 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. 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 with Tris-EDTA buffer citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling DMC1 with ab245217 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on spermatocytes of rat testis. The section was incubated with ab245217 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. 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 with Tris-EDTA buffer citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling DMC1 with ab245217 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on spermatocytes of mouse testis. The section was incubated with ab245217 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. 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 with Tris-EDTA buffer citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins.