Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed 0.1% TritonX-100 permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) cells labelling TRPML1/MG-2 with ab323642 at 1/100 (5.07 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing lysosomal staining in PC-3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Low expression : Ramos

ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain tubulin at 1/ab25631 50 2ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ab150081 1000 2ug/ml dilution.

-ve control 1 : ab323642 at 1/100 dilution followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.-ve control 2 : ab25631 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling TRPML1/MG-2 with ab323642 at 1/500 (1.014 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse liver. The section was incubated with ab323642 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling TRPML1/MG-2 with ab323642 at 1/100 (5.07 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing lysosomal staining in Neuro-2a cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Low expression : F9

ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain tubulin at 1/50 2ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ab150081 1000 2ug/ml dilution.

-ve control 1 : ab323642 at 1/100 dilution followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.-ve control 2 : ab25631 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded (A) Neuro-2a (mouse neuroblastoma neuroblast) cell pellet (B) F9 (mouse embryonal carcinoma epithelial cell) cell pellet tissue labeling TRPML1/MG-2 with ab323642 at 1/500 (1.014 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Neuro-2a cell pellet no staining on (B) F9 cell pellet. The section was incubated with ab323642 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling TRPML1/MG-2 with ab323642 at 1/500 (1.014 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum. The section was incubated with ab323642 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins

• WB

Supplier Data

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

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

Negative control : Ramos.

BFA treatment can prevent cleavage (PMID : 16517607).

20-37(cleaved form) 60-75 (full length) 120-150(dimer form) 250-300 (oligermer). The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16517607).

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

In Western blot Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] (<a href='/en-us/products/primary-antibodies/trpml1-mg-2-antibody-epr28651-127-ab323642'>ab323642</a>) at 1/1000 dilution

Lane 1:

Untreated PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

PC-3 treated with 300ng/ml BFA for 24 hours whole cell lysate at 50 µg

Lane 3:

Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 20-37 kDa,60-75 kDa,120-150 kDa,250-300 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using [Standard ab323642], the same antibody clone in a different buffer formulation.

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

In Western blot Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16517607).

Observed MW (kDa) : 20-37 (cleaved form) 60-75 (full length).

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

All lanes:

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] (<a href='/en-us/products/primary-antibodies/trpml1-mg-2-antibody-epr28651-127-ab323642'>ab323642</a>) at 1/1000 dilution

Lane 1:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

PC-3 transfected with siRNA specifically targeting TRPML1/MG-2 whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 20-37 kDa,60-75 kDa

false

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

Dot blot analysis of TRPML1/MG-2 using ab323642 at 1000 (0.507 ug/ml) followed by a Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated (ab97051) at 1 : 100000 dilution.

Lane1 : Human MCOLN1 peptide
Lane2 : Human TAF1C peptide

Exposure time : 180 seconds.

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

This antibody does not cross-react with TAF1C.

All lanes:

Dot Blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] (<a href='/en-us/products/primary-antibodies/trpml1-mg-2-antibody-epr28651-127-ab323642'>ab323642</a>) at 1/1000 dilution

Lane 1:

Human MCOLN1 peptide

Lane 2:

Human TAF1C peptide

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

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

Low expression : F9.

BFA treatment can prevent cleavage (PMID : 16517607).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16517607).

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

In Western blot Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] (<a href='/en-us/products/primary-antibodies/trpml1-mg-2-antibody-epr28651-127-ab323642'>ab323642</a>) at 1/1000 dilution

Lane 1:

Untreated Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 80 µg

Lane 2:

Neuro-2a treated with 300ng/ml BFA for 24 hours whole cell lysate at 80 µg

Lane 3:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 80 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 20-37 kDa,60-75 kDa,120-150 kDa,250-300 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] - BSA and Azide free (AB325960)

This data was developed using ab323642, the same antibody clone in a different buffer formulation.

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

This antibody does not cross-react with human MCOLN3.

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

In Western blot Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

Loading control : ab181602 Anti-GAPDH antibody [EPR16891] at 1/200000 dilution and ab213204 Anti-6X His tag® antibody [EPR20547] - ChIP Grade at 1/5000 dilution.

All lanes:

Western blot - Anti-TRPML1/MG-2 antibody [EPR28651-127] (<a href='/en-us/products/primary-antibodies/trpml1-mg-2-antibody-epr28651-127-ab323642'>ab323642</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human MCOLN3 expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human MCOLN1 expression vector containing a His-tag, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 20-37 kDa,60-75 kDa,120-150 kDa,250-300 kDa,36 kDa,20-300 kDa

false

Exposure time: 3s