Anti-GAPDH antibody [EPR16891] - Loading Control

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocyte of rat spleen is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling GAPDH with ab181602 at 1/180 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized HeLa (human cervix adenocarcinoma) cells labeling GAPDH with ab181602 at dilution of 1/500. The secondary antibody used was Alexa Fluor^® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/400. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/500) was used to stain tubulin along with ab150120 (AlexaFluor^®594 goat anti-mouse secondary, 1/500). The negative controls are shown in the bottom middle and right hand panels- for negative control 1 primary antibody (ab181602; 1/500) and secondary antibody (ab150120; 1/500) was used. For negative control 2 primary antibody (ab7291; 1/500) and secondary antibody (ab150077; 1/400) was used.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded human transitional cell carcinoma of bladder tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic and nucleus staining on the tumor cells of transitional cell carcinoma of human bladder is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

STT3A was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate with ab320831 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320831 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

In Immunoprecipitation, ab320831 was shown to bind specifically to STT3A. Target of interest was enriched at 60 kDa in wild-type HEK-293T cell lysates (lane 2) with no signal observed at this size in STT3A knockout cell line (lane 5) (lane 5, knockout cell line ab266320 / knockout cell lysate ab259164).
The bands above 60kDa are likely to be aggregation, and are absent in the KO samples. Unlike the Western Blot data, these samples have been boiled. ab181602 was used as a GAPDH loading control.

All lanes:

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] (<a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in Parental HEK-293T whole cell lysate

Lane 4:

STT3A KO HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 5:

Immunoprecipitation - Human STT3A knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-stt3a-knockout-hek-293t-cell-line-ab266320'>ab266320</a>)

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in STT3A KO HEK-293T whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 60 kDa

false

Exposure time: 84s

• IP

Supplier Data

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Dnmt3a was immunoprecipitated from 0.35 mg Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab323708 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323708 at 1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-Dnmt3a antibody [EPR29184-89] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a>) at 1/1000 dilution

Lane 1:

(Input) Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> at 1/30 IP in Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> in parental HeLa whole cell lysate at 10 µg

Lane 4:

(Input) DNMT3A knockout HeLa whole cell lysate at 10 µg

Lane 5:

<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> at 1/30 IP in DNMT3A knockout HeLa whole cell lysate at 10 µg

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> in DNMT3A knockout HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 130 kDa,36 kDa

false

Exposure time: 24s

• IP

Supplier Data

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

GAPDH was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab181602 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab181602 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : HeLa whole cell extract.

Lane 2 : PBS instead of HeLa whole cell extract.

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

All lanes:

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602)

Predicted band size: 36 kDa

Observed band size: 36 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocytes of mouse spleen is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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 : 21878523, PMID : 20949559; PMID : 28106765).

The identity of the bands lower than 130 kDa in lanes 1-4 are unknown.

Lanes 1-4 are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and lanes 5-7 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-ECE1 antibody [EPR30300-555] (<a href='/en-us/products/primary-antibodies/ece1-antibody-epr30300-555-ab326103'>ab326103</a>) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human lung tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Lane 4:

Human colon cancer tissue lysate at 20 µg

Lane 5:

Mouse placenta tissue lysate at 20 µg

Lane 6:

Mouse liver tissue lysate at 20 µg

Lane 7:

Mouse lung tissue lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 5 - 7:

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: 130 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : HeLa.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the lower MW band at approximately 60 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-INPP5F antibody [MJF-D29759-51] (<a href='/en-us/products/primary-antibodies/inpp5f-antibody-mjf-d29759-51-ab325711'>ab325711</a>) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Performed under reducing conditions.

In Western blot, ab326929 was shown to bind specifically to Dishevelled 2. Target of interest was observed at 95 kDa in wild-type A549 cell lysates (lane 3) with no signal observed at this size in Dishevelled 2 knockout cell line (lane 4) (knockout cell line ab277903 / knockout cell lysate ab288317).

In lane 4, the lysate was stored at -80℃ prior to Western Blotting. The bands beneath the target band (95 kDa) are likey to be degradation products. In lanes 1-3, To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-4 : 59 seconds.

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (<a href='/en-us/products/primary-antibodies/dishevelled-2-antibody-epr30298-73-ab326929'>ab326929</a>) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Dishevelled 2 knockout A549 whole cell lysate at at 20 µg

Lanes 3 - 4:

HeLa (human cervical adenocarcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 95 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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 : 14762142).

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

The identity of the bands between 25 kDa and 37 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-KCNQ2 antibody [EPR29531-83] (<a href='/en-us/products/primary-antibodies/kcnq2-antibody-epr29531-83-ab325971'>ab325971</a>) at 1/1000 dilution

Lane 1:

Untreated SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

SH-SY5Y treated with 10 uM retinoic acid for 7 days, 50ng/ml BDNF was then added for additional 7 days whole cell lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 100 kDa,75 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

ab181602 was used as GAPDH loading control at a 1/2200000 dilution. ab201456 was used for total protein control.

All lanes:

Western blot - Anti-Histone H3 (phospho S28) antibody [HTA28] (<a href='/en-us/products/primary-antibodies/histone-h3-phospho-s28-antibody-hta28-ab10543'>ab10543</a>) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

HeLa treated with 100ng/ml nocodazole for 16 hours whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated HeLa whole cell lysate (Lambda Protein Phosphatase treated membrane) at 20 µg

Lane 4:

HeLa treated with 100ng/ml nocodazole for 16 hours whole cell lysate (Lambda Protein Phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot at 1/10000 dilution

Predicted band size: 15 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : THP-1.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the lower MW band at approximately 60 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-INPP5F antibody [MJF-D29759-51] (<a href='/en-us/products/primary-antibodies/inpp5f-antibody-mjf-d29759-51-ab325711'>ab325711</a>) at 1/1000 dilution

Lane 1:

SK-MEL-28 (human malignant melanoma cell) whole cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia monocyte) 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 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

All lanes:

Western blot - Anti-ATP4A antibody [EPR12251] (<a href='/en-us/products/primary-antibodies/atp4a-antibody-epr12251-ab174293'>ab174293</a>) at 1/1000 dilution

Lane 1:

Human stomach tissue lysate at 20 µg

Lane 2:

Mouse stomach tissue lysate at 20 µg

Lane 3:

Rat stomach tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 114 kDa,36 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : liver.

The bands beneath the target band (532 kDa) are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-DYNC1H1 antibody [EPR30299-93] (<a href='/en-us/products/primary-antibodies/dync1h1-antibody-epr30299-93-ab325710'>ab325710</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

Hela transfected with siRNA specifically targeting DYNC1H1 whole cell lysate at 20 µg

Lane 3:

Human cerebellum tissue lysate at 20 µg

Lane 4:

Human testis tissue lysate at 20 µg

Lane 5:

Human liver tissue 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: 532 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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 : 29478807, 11470414). The molecular weight of ASF1A is upshifted when phosphorylated.

To minimize protein degradation, the fresh lysates were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the bands higher than 37 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-ASF1B (phospho S198) antibody [EPR28822-77] (<a href='/en-us/products/primary-antibodies/asf1b-phospho-s198-antibody-epr28822-77-ab325649'>ab325649</a>) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 48 µg

Lane 2:

HeLa treated with 100ng/ml Calyculin A for 30 min whole cell lysate at 48 µg

Lane 3:

HeLa transfected with scrambled siRNA control treated with 100ng/ml Calyculin A for 30 min whole cell lysate at 48 µg

Lane 4:

HeLa transfected with siRNA specifically targeting ASF1B treated with 100ng/ml Calyculin A for 30 min whole cell lysate at 48 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: 18 kDa,20 kDa,36 kDa

false

Exposure time: 147s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : U-118 MG.

The identity of the bands lower than 60 kDa are unknown.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD2AP antibody [EPR30265-529] (<a href='/en-us/products/primary-antibodies/cd2ap-antibody-epr30265-529-ab325800'>ab325800</a>) at 1/1000 dilution

Lane 1:

HEL (human erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 2:

U-118 MG (human brain glioblastoma cell) 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 1/20000 dilution

Observed band size: 80 kDa,36 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

In Western blot, ab326338 was shown to bind specifically to KEAP1.
Target of interest was observed at 70 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in HAP1 knockout cell line (lane 2).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (<a href='/en-us/products/primary-antibodies/kelch-like-ech-associated-protein-1-antibody-epr31136-529-ab326338'>ab326338</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP-1 (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate at 60 µg

Lane 2:

KEAP1 knockout HAP1 whole cell lysate at 60 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 60 µg

Lane 4:

Calu-1 (human lung epidermoid carcinoma epithelial cell) whole cell lysate at 60 µg

Lane 5:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 60 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: 70 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

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

All lanes:

Western blot - Anti-Notch1 antibody [EP1238Y] (<a href='/en-us/products/primary-antibodies/notch1-antibody-ep1238y-ab52627'>ab52627</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell)

Lane 2:

293T (human embryonic kidney epithelial cell)

Lane 3:

NIH/3T3 (mouse embryonic fibroblast)

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: 110 kDa,272 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

The identity of the bands higher than 100 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-ASF1B (phospho S198) antibody [EPR28822-77] (<a href='/en-us/products/primary-antibodies/asf1b-phospho-s198-antibody-epr28822-77-ab325649'>ab325649</a>) at 1/1000 dilution

Lane 1:

Untreated Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Raji treated with 100ng/ml Calyculin A for 30 min 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 1/20000 dilution

Observed band size: 18 kDa,20 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CISD2 antibody [EPR30277-16] (<a href='/en-us/products/primary-antibodies/cisd2-antibody-epr30277-16-ab325526'>ab325526</a>) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HEK-293 transfected with siRNA specifically targeting CISD2 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: 15 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : K-562, PANC-1

The bands beneath the target band (51 kDa) are likely to be degraded target fragments.

Lane 1 of this blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lane 1 : 125 seconds; Lanes 2-4 : 180 seconds

All lanes:

Western blot - Anti-OTUD1 antibody [EPR30462-31] (<a href='/en-us/products/primary-antibodies/otud1-antibody-epr30462-31-ab326105'>ab326105</a>) at 1/1000 dilution

Lane 1:

AsPC-1 (human Pancreas epithelial cell) whole cell lysate at 48 µg

Lane 2:

U266B1 (human multiple myeloma B lymphocyte) whole cell lysate at 48 µg

Lane 3:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 48 µg

Lane 4:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 48 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: 51 kDa,36 kDa

true

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.
ab181602 was used as a loading control at a 1/200000 dilution.

The expression of PINK1 is upregulated in response to valinomycin treatment (PMID : 22724072).

Left panel is incubated with Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/2000 and right panel is incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000.

The blot of left panel was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

The blot of right panel was developed using a ECL substrate, allowing for the detection of proteins in the picogram to high-femtogram range.

All lanes:

Western blot - Anti-PINK1 antibody [N4/15] (<a href='/en-us/products/primary-antibodies/pink1-antibody-n4-15-ab186303'>ab186303</a>) at 1/1000 dilution

Lane 1:

Untreated SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate at 20 µg

Lane 2:

SK-OV-3 treated with 30μM CCCP for 6h, 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 1/2000 dilution

Predicted band size: 62 kDa

true

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

The bands beneath the target band (51 kDa) are likely to be degraded target fragments.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-OTUD1 antibody [EPR30462-31] (<a href='/en-us/products/primary-antibodies/otud1-antibody-epr30462-31-ab326105'>ab326105</a>) at 1/1000 dilution

Lane 1:

KARPAS-299 (human T cell lymphoma cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

KARPAS-299 transfected with siRNA specifically targeting OTUD1 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 1/20000 dilution

Observed band size: 51 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : colon (PMID : 11741232, PMID : 11741260)

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 33560415; PMID : 37527038).

This blot of lanes1-4 and lane 7 was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

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

The identity of the higher MW band at approximately 300 kDa in lane 7 is unknown.

The identity of the lower MW band at approximately 40 kDa in lane 3 is unknown.

Lanes 1-4 and lane 7 are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and lanes 5-6 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000.

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

Exposure time : Lanes 1-4 : 48 seconds; Lanes 6-7 : 180 seconds

All lanes:

Western blot - Anti-SLC15A4/PHT1 antibody [EPR26040-123] (<a href='/en-us/products/primary-antibodies/slc15a4-pht1-antibody-epr26040-123-ab324433'>ab324433</a>) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 48 µg

Lane 2:

Human liver tissue lysate at 48 µg

Lane 3:

Human tonsil tissue lysate at 48 µg

Lane 4:

Human colon tissue lysate at 48 µg

Lane 5:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 48 µg

Lane 6:

A-673 (human muscle Ewings sarcoma cell line) whole cell lysate at 48 µg

Lane 7:

Human skeletal muscle tissue lysate at 48 µg

Secondary

Lanes 1, 2, 3, 4 and 7:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 5 - 6:

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: 50-100 kDa,36 kDa

true

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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 : 33560415; PMID : 37527038).

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

The identity of the lower MW band at approximately 40 kDa in lane 1 and lane 3 is unknown.

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

All lanes:

Western blot - Anti-SLC15A4/PHT1 antibody [EPR26040-123] (<a href='/en-us/products/primary-antibodies/slc15a4-pht1-antibody-epr26040-123-ab324433'>ab324433</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole lysate at 40 µg

Lane 2:

293T mitochondrial fraction at 40 µg

Lane 3:

293T non-mitochondrial fraction 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 1/20000 dilution

Observed band size: 50-100 kDa,36 kDa,16 kDa

false

Exposure time: 136s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

ab181602 was used as a GAPDH loading control at a 1/200000 dilution.

Low expression : HepG2
Negative control : SW480.

All lanes:

Western blot - Anti-Viperin antibody [MaP.VIP] (<a href='/en-us/products/primary-antibodies/viperin-antibody-mapvip-ab107359'>ab107359</a>) at 1/1000 dilution

Lane 1:

HCC1937 (human mammary gland epithelial cell) whole cell lysate at 20 µg

Lane 2:

Calu-3 (human lung adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

KG-1a (human bone marrow myeloblast) whole cell lysate at 20 µg

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 7:

RAW264.7 treated with 100ng/ml LPS and 100U/ml IFN gamma for 4h whole cell lysate at 20 µg

Lane 8:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 9:

THP-1 treated with 100ng/ml LPS and 100U/ml IFN gamma for 4h whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Observed band size: 42 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : HT-29, HL-60.

The molecular weight observed is consistent with what has been described in the literature (PMID : 22227581; PMID : 12006591).

Bands observed at around 250 kDa may represent oligomeric forms of MMP11.

The identity of the lower MW band at approximately 20 kDa in lane 6 is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-MMP11 antibody [EPR29490-537R] (<a href='/en-us/products/primary-antibodies/mmp11-antibody-epr29490-537r-ab326424'>ab326424</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 48 µg

Lane 2:

HepG2 culture supernatant (ultrafiltration) at 48 µg

Lane 3:

TT (human thyroid carcinoma epithelial cell) whole cell lysate at 48 µg

Lane 4:

TT culture supernatant (ultrafiltration) at 48 µg

Lane 5:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg

Lane 6:

HT-29 culture supernatant (ultrafiltration) at 48 µg

Lane 7:

HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 48 µg

Lane 8:

HL-60 culture supernatant (ultrafiltration) at 48 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: 55 kDa,44 kDa,37 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the lower MW band at approximately 60 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-INPP5F antibody [MJF-D29759-51] (<a href='/en-us/products/primary-antibodies/inpp5f-antibody-mjf-d29759-51-ab325711'>ab325711</a>) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

A375 transfected with siRNA specifically targeting INPP5F 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 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 169s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Negative control : kidney, spleen, liver, testis

Lanes 1-3 are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and lanes 4-9 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Stathmin-2/STMN2 antibody [EPR31028-544] (<a href='/en-us/products/primary-antibodies/stathmin-2-stmn2-antibody-epr31028-544-ab326937'>ab326937</a>) at 1/1000 dilution

Lane 1:

Human cerebral cortex tissue lysate at 20 µg

Lane 2:

Human kidney tissue lysate at 20 µg

Lane 3:

Human spleen tissue lysate at 20 µg

Lane 4:

Mouse brain tissue lysate at 20 µg

Lane 5:

Mouse liver tissue lysate at 20 µg

Lane 6:

Mouse testis tissue lysate at 20 µg

Lane 7:

Rat brain tissue lysate at 20 µg

Lane 8:

Rat liver tissue lysate at 20 µg

Lane 9:

Rat testis tissue lysate at 20 µg

Secondary

Lanes 1 - 3:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 4 - 9:

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 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

To minimize protein degradation, cells (lanes 5-8) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-5 : 59 seconds; Lanes 6-8 : 180 seconds

All lanes:

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR29602-146] (<a href='/en-us/products/primary-antibodies/kdm4a-jhdm3a-jmjd2a-antibody-epr29602-146-ab326104'>ab326104</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

3T3-L1 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 5:

Mouse spleen tissue lysate at 20 µg

Lane 6:

HeLa (human cervical adenocarcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 7:

293T (human embryonic kidney epithelial cell) fresh whole cell lysate at 20 µg

Lane 8:

NIH/3T3 (mouse embryonic fibroblast) fresh 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 1/20000 dilution

Observed band size: 150 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

In Western blot, ab326104 was shown to bind specifically to KDM4A / JHDM3A / JMJD2A. Target of interest was observed at 150 kDa in wild-type HPA1 cell lysates (lane 1) with no signal observed at this size in KDM4A / JHDM3A / JMJD2A knockout cell line.

To minimize protein degradation, tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-KDM4A / JHDM3A / JMJD2A antibody [EPR29602-146] (<a href='/en-us/products/primary-antibodies/kdm4a-jhdm3a-jmjd2a-antibody-epr29602-146-ab326104'>ab326104</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

KDM4A / JHDM3A / JMJD2A knockout HAP1 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: 150 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

The identity of the higher MW band at approximately 150 kDa (in lane 2) is unknown.

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

In Western blot, Anti-RPS6KB1 antibody [E343] (ab32529) staining at 1/1000 dilution.

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

20-kDa mature form of Cathepsin C is observed. The MW is consistent with that described in the literature (PMID : 25244098).

In Western blot, ab314644 was shown to bind specifically to Cathepsin C.

Target of interest was observed at 20 kDa in wild-type HeLa cell lysates (lane 1, wild-type cell line ab255928) with no signal observed at this size in CTSC knockout cell line (lane 2, knockout cell line ab265822/ knockout cell lysate ab257909). In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-NELFe antibody [EPR29850-562] (ab326107) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

293T transfected with siRNA specifically targeting NELFe 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 1/20000 dilution

Observed band size: 43 kDa,36 kDa

false

Exposure time: 125s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Negative control : U-2 OS, 293T, NIH/3T3

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Stathmin-2/STMN2 antibody [EPR31028-544] (<a href='/en-us/products/primary-antibodies/stathmin-2-stmn2-antibody-epr31028-544-ab326937'>ab326937</a>) at 1/1000 dilution

Lane 1:

SK-N-BE(2) (human neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 2:

SK-N-SH (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 6:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma cell) 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 1/20000 dilution

Observed band size: 20 kDa,36 kDa

false

Exposure time: 136s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : SK-N-BE(2), lung, skeletal muscle

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 26 seconds, lane 3 : 92 seconds, lanes 4-5 : 103 seconds

All lanes:

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (<a href='/en-us/products/primary-antibodies/atp13a2-antibody-mjf-d29756-28-ab322735'>ab322735</a>) at 1/1000 dilution

Lane 1:

Saos-2 (human osteosarcoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

SK-N-BE(2) (human neuroblastoma neuroblast) whole cell lysate at 50 µg

Lane 3:

Human cerebellum tissue lysate at 50 µg

Lane 4:

Human hypothalamus tissue lysate at 50 µg

Lane 5:

Human skeletal muscle tissue lysate at 50 µg

Lane 6:

Human lung muscle tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 150 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

In Western blot analysis, ab233706 exhibited lower sensitivity compared to ab233551, for which reason we recommend the latter one as superior alternative.

ab233706 does not work in THP-1 cell line due to sensitivity issue.

Lanes 1 - 2:

Western blot - Anti-IL-6 antibody [EPR21711] (<a href='/en-us/products/primary-antibodies/il-6-antibody-epr21711-ab233706'>ab233706</a>) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-IL-6 antibody [EPR22565-204] (<a href='/en-us/products/primary-antibodies/il-6-antibody-epr22565-204-ab233551'>ab233551</a>) at 1/1000 dilution

Lanes 1 and 3:

Untreated HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Lanes 2 and 4:

HUVEC treated with 0.5 ug/ml lipopolysaccharides (LPS) for 24 hours, added 300 ng/ml BFA last 20 hours 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 1/20000 dilution

Observed band size: 23 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

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

Low expression : K-562.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SH3MD2 antibody [EPR30521-594] (<a href='/en-us/products/primary-antibodies/sh3md2-antibody-epr30521-594-ab326568'>ab326568</a>) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 2:

K-562 (human chronic myelogenous leukemia lymphoblast) fresh 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 1/20000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 147s