All lanes : Anti-SYT2 + SYT1 antibody [EPR23920-2] (ab259977) at 1/1000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human liver tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 47 kDa
Observed band size: 60/37 kDa why is the actual band size different from the predicted?

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

Unknown band at 37 kDa (PMID:32930942).

Negative: human liver (PMID:17492637).

Exposure time: 5.5 seconds

 

All lanes : Anti-SYT2 + SYT1 antibody [EPR23920-2] (ab259977) at 1/1000 dilution

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 2 : C57 spinal cord tissue lysate
Lane 3 : Mouse brain tissue lysate
Lane 4 : Mouse liver tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 47 kDa
Observed band size: 60/48/37 kDa why is the actual band size different from the predicted?

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

Unknown band at 37 kDa (PMID:32930942).

Negative: mouse liver (PMID:17492637).

Exposure time: Lane 1, 2, 4: 10 secondsLane 3: 3.3 seconds

 

All lanes : Anti-SYT2 + SYT1 antibody [EPR23920-2] (ab259977) at 1/1000 dilution

Lane 1 : His-tagged mouse recombinant protein SYT1 (aa137-421) (35 kDa)
Lane 2 : His-tagged mouse recombinant protein SYT2 (aa137-420) (34.8 kDa)
Lane 3 : His-tagged mouse recombinant protein SYT3 (aa 76-587) (56.9 kDa)
Lane 4 : His-tagged mouse recombinant protein SYT4 (aa 38-425) (45.6 kDa)
Lane 5 : His-tagged mouse recombinant protein SYT5 (aa 46-386) (40.4 kDa)
Lane 6 : His-tagged mouse recombinant protein SYT6 (aa 81-511) (51kDa)
Lane 7 : His-tagged mouse recombinant protein SYT7 (aa 38-403) (43.6 kDa)
Lane 8 : His-tagged mouse recombinant protein SYT9 (aa 74-491) (50.2 kDa)

Lysates/proteins at 0.03 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: 47 kDa
Observed band size: 34.8-56.9 kDa why is the actual band size different from the predicted?

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

This antibody reacts with mouse SYT1 and SYT2 but not with SYT3, SYT4, SYT5, SYT6, SYT7, SYT9. These rec proteins were both made in house and extracted from E.coil expression systems.

Exposure time: 15 seconds

 

Immunohistochemical analysis of paraffin-embedded Mouse cerebellum tissue labeling SYT1 + SYT2 with ab259977 at 1/50 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse cerebellum. The section was incubated with ab259977 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 cerebellum tissue labeling SYT1 + SYT2 with ab259977 at 1/50 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat cerebellum (PMID: 1856191). The section was incubated with ab259977 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 lung tissue labeling SYT1 + SYT2 with ab259977 at 1/50 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on mouse lung (PMID: 1856191). The section was incubated with ab259977 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 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse hippocampus tissue labeling SYT1 + SYT2 with ab259977 at 1/50 dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Positive staining on mouse hippocampus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling SYT1 + SYT2 with ab259977 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing positive staining in synapse of mouse primary neurons. Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection is observed. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

SYT1 + SYT2 was immunoprecipitated from 0.35 mg Human cerebellum tissue lysate 10 ug with ab259977 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259977 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Human cerebellum tissue lysate 10 ug

Lane 2: ab259977 IP in Human cerebellum tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259977 in human cerebellum tissue lysate

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

Exposure time: 3 minutes