Recombinant Anti-VMAT2 antibody [EPR24197-51] (ab259970)

All lanes : Anti-VMAT2 antibody [EPR24197-51] (ab259970) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse hippocampus tissue lysate
Lane 3 : Mouse spleen tissue lysate
Lane 4 : Mouse skin tissue lysate
Lane 5 : Mouse thyroid tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1 & 3-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Lane 2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 56 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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

Samples are non-boiled as boiling may cause protein aggregates. 

This protein is specifically expressed in CNS.

Exposure time: 48 seconds

 

All lanes : Anti-VMAT2 antibody [EPR24197-51] (ab259970) at 1/1000 dilution

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat hippocampus tissue lysate
Lane 3 : Rat spleen tissue lysate
Lane 4 : Rat skin tissue lysate
Lane 5 : Rat thyroid tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 56 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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

Samples are non-boiled as boiling may cause protein aggregates. 

This protein is specifically expressed in CNS.

Exposure time: Lane 1: 29 seconds

Lane 2-5: 59 seconds

 

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum (PMID: 27836486). The section was incubated with ab259970 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 (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum (PMID: 27836486). The section was incubated with ab259970 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 (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum. The section was incubated with ab259970 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 (pH 9.0, epitope retrieval solution 2) for 20 minutes.

VMAT2 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate 10 ug with ab259970 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259970 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10 ug

Lane 2: ab259970 IP in Mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259970 in Mouse brain tissue lysate

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

Exposure time: 10 seconds

Input sample is non-boiled as boiling may cause protein aggregates.

VMAT2 was immunoprecipitated from 0.35 mg Rat brain tissue lysate 10 ug with ab259970 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259970 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Rat brain tissue lysate 10 ug

Lane 2: ab259970 IP in Rat brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259970 in Rat brain tissue lysate

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

Exposure time: 10 seconds

Input sample is non-boiled as boiling may cause protein aggregates.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: almost no staining on mouse spleen.The section was incubated with ab259970 for 30 minutes 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 (pH 9.0, epitope retrieval solution2) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Mouse skin tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: almost no staining on mouse skin.The section was incubated with ab259970 for 30 minutes 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 (pH 9.0, epitope retrieval solution2) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: almost no staining on rat spleen. The section was incubated with ab259970 for 30 minutes 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 (pH 9.0, epitope retrieval solution2) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Rat skin tissue labeling VMAT2 with ab259970 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: almost no staining on rat skin. The section was incubated with ab259970 for 30 minutes 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 (pH 9.0, epitope retrieval solution2) for 20 minutes.

Immunofluorescence staining of VMAT2 using ab259970 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab259970 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

Immunofluorescence staining of VMAT2 using ab259970 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab259970 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.