Goat Anti-Rabbit IgG H&L (HRP)

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-RAB29 (phospho T71) antibody [MJF-R24-17-1] (<a href='/en-us/products/primary-antibodies/rab29-phospho-t71-antibody-mjf-r24-17-1-ab241062'>ab241062</a>) at 1/5000 dilution

Lane 1:

HEK-293 (human embryonic kidney) over-expressing HA-tagged RAB29 and LRRK2 [Y1699C], whole cell lysate at 15 µg

Lane 2:

MLi-2 (100nM for 60min) treated HEK-293 over-expressing HA-tagged RAB29 and LRRK2 [Y1699C], whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 23 kDa

Observed band size: 23 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Immunohistochemical analysis of paraffin-embedded Human skin tissue labeling Histone H3 (mono methyl K79) with ab177183 at 1/5000 dilution, followed by ab97051 Goat Anti-Rabbit HRP (IgG H&L) at 1/500 dilution. Nucleus staining on Human skin tissue 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Immunohistochemical staining of paraffin embedded human endometrial carcinoma with purified ab32063 at a working dilution of 1 in 200. The secondary antibody used is ab97051 a HRP goat anti-rabbit IgG (H+L) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer pH 9.0.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

ab92511 staining HLA-DR in human spleen tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/700. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500. Negative control 1 : PBS in place of primary antibody.

• IP

Supplier Data

Immunoprecipitation - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

DDR1 (phospho Y796) + DDR2 (phospho Y740) was immunoprecipitated from 0.35 mg T-47D treated with 20 ug/ml Collagen I for 18 hours whole cell lysate with ab322921 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322921 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-DDR1 (phospho Y796) + DDR2 (phospho Y740) antibody [EPR28699-33] (<a href='/en-us/products/primary-antibodies/ddr1-phospho-y796-ddr2-phospho-y740-antibody-epr28699-33-ab322921'>ab322921</a>) at 1/1000 dilution

Lane 1:

T-47D treated with 20 ug/ml Collagen I for 18 hours whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/ddr1-phospho-y796-ddr2-phospho-y740-antibody-epr28699-33-ab322921'>ab322921</a> at 1/30 IP in T-47D treated with 20 ug/ml Collagen I for 18 hours 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/ddr1-phospho-y796-ddr2-phospho-y740-antibody-epr28699-33-ab322921'>ab322921</a> in T-47D treated with 20 ug/ml Collagen I for 18 hours whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/5000 dilution

Observed band size: 125 kDa

false

Exposure time: 76s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

This data was developed using ab259869, the same antibody clone in a different buffer formulation.

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

This antibody may react with an unidentifiable protein around 30 kDa.

Exposure time : 62 seconds

All lanes:

Western blot - Anti-DYRK1A + DYRK1B antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

DYRK1A knockdown HeLa whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Low expression : AsPC-1, K-562, U-937.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

All lanes:

Western blot - Anti-Integrin alpha 1/CD49a antibody [EPR30388-592] - BSA and Azide free (ab326330) at 1/1000 dilution

Lane 1:

Huh7 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

AsPC-1 (human Pancreas epithelial cell) whole cell lysate at 20 µg

Lane 5:

U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 230 kDa,124 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Negative control : COLO 201.

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

All lanes:

Western blot - Anti-c-Myb antibody [EPR27202-86] (<a href='/en-us/products/primary-antibodies/c-myb-antibody-epr27202-86-ab324970'>ab324970</a>) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 80 µg

Lane 2:

COLO 201 (human colon adenocarcinoma tumor ascitic fibroblast-like cell) whole cell lysate at 80 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 75 kDa,36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa);Anti-Ret antibody (ab134100) (1 : 1000) (150KDa).

All lanes:

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/1000 dilution

Lane 1:

Untreated TT (human thyroid carcinoma epithelial cell) whole cell lysate 20 µg (untreated membrane)

Lane 2:

TT treated with 100uM pervanadate for 10 min whole cell lysate 20 µg (untreated membrane)

Lane 3:

Untreated TT (human thyroid carcinoma epithelial cell) whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 4:

TT treated with 100uM pervanadate for 10 min whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 150 kDa,36 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

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

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

All lanes:

Western blot - Anti-Plasmolipin antibody [EPR31074-536] (ab325932) at 1/1000 dilution

Lane 1:

Human stomach tissue lysate at 20 µg

Lane 2:

Human kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/2000 dilution

Observed band size: 20 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD27 antibody [EPR8569] - Low endotoxin, Azide free (<a href='/en-us/products/primary-antibodies/cd27-antibody-epr8569-low-endotoxin-azide-free-ab192336'>ab192336</a>)

All lanes:

Human spleen lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 29 kDa

Observed band size: 55 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

The expression of NLRC5 is upregulated in response to IFN gamma treatment (PMID : 36103539; PMID : 20610642; PMID : 20061403).

This antibody can recognize the different isoforms of NLRC5.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

All lanes:

Western blot - Anti-NLRC5 antibody [EPR29722-55] (<a href='/en-us/products/primary-antibodies/nlrc5-antibody-epr29722-55-ab325273'>ab325273</a>) at 1/1000 dilution

Lane 1:

Untreated Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 2:

Jurkat treated with 10ng/ml IFN gamma for 24 hours whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

THP-1 treated with 10ng/ml IFN gamma for 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 205 kDa,170 kDa,124 kDa

false

Exposure time: 114s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

The expression of NLRC5 is upregulated in response to IFN gamma treatment (PMID : 36103539; PMID : 20610642; PMID : 20061403).

This antibody can recognize the different isoforms of NLRC5.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

All lanes:

Western blot - Anti-NLRC5 antibody [EPR29722-55] (<a href='/en-us/products/primary-antibodies/nlrc5-antibody-epr29722-55-ab325273'>ab325273</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

HeLa treated with 1000U/ml IFN gamma for 6 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 205 kDa,170 kDa,124 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

This data was developed using ab259869, the same antibody clone in a different buffer formulation.

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

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 70 seconds

All lanes:

Western blot - Anti-DYRK1A + DYRK1B antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix 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:

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

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 11483509, PMID : 33483465).

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

All lanes:

Western blot - Anti-Cathepsin L/MEP antibody [EPR30887-518] (ab326362) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 3:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 5:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 6:

Human liver tissue lysate at 40 µg

Lane 7:

Human kidney tissue lysate at 40 µg

Lane 8:

Human spleen tissue lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 38 kDa,30 kDa,25 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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-AGO2(phospho S387) antibody [EPR29226-168] (ab326569) at 1/1000 dilution

Lane 1:

HEK293T cells were co-transfected with human AGO2(S387A) (His-tagged) and human AKT3 (EGFP-tagged) expression vectors, an upstream kinase used to enhance AGO2 phosphorylation at S387, whole cell lysate 20 µg (Untreated membrane)

Lane 2:

HEK293T cells were co-transfected with human AGO2 (His-tagged) and human AKT3 (EGFP-tagged) expression vectors, an upstream kinase used to enhance AGO2 phosphorylation at S387, whole cell lysate 20 µg (Untreated membrane)

Lane 3:

HEK293T cells were co-transfected with human AGO2 (His-tagged) and human AKT3 (EGFP-tagged) expression vectors, an upstream kinase used to enhance AGO2 phosphorylation at S387, whole cell lysate 20 µg (Alkaline phosphatase treated membrane)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 97 kDa,36 kDa,75 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : HCT116, HEK-293 (PMID : 23743647). The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 11021599). In lane 1, the lysate was stored at -80°C prior to Western Blotting. The bands beneath the target band (320 kDa) are likey to be degradation products. In lanes 2-4, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation. In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.

All lanes:

Western blot - Anti-Collagen VII antibody [EPR27012-157] (<a href='/en-us/products/primary-antibodies/collagen-vii-antibody-epr27012-157-ab309143'>ab309143</a>) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 320 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Performed under reducing conditions. In Western blot, ab307548 was shown to bind specifically to SIGMAR1. A band was observed at 26 kDa in wild-type 293T cell lysates with whereas no signal observed at this size in SIGMAR1 knockout cell line ab266619 (knockout cell lysate ab258666). Exposure time : Lanes 1-2 : 180 seconds; Lane 3 : 136 seconds.

All lanes:

Western blot - Anti-Sigma1-receptor antibody [EPR25145-11] (<a href='/en-us/products/primary-antibodies/sigma1-receptor-antibody-epr25145-11-ab307548'>ab307548</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

SIGMAR1 knockout HEK293T whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

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

Lane 6:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 26 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Blocking and diluting buffer and concentration : 5% NFDM /TBST.
DYRK1 has been shown to undergo degradation (PMID : 27807027)

Lanes 1 - 3:

Western blot - Anti-DYRK1A + DYRK1B antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-mCherry antibody [EPR20579] (<a href='/en-us/products/primary-antibodies/mcherry-antibody-epr20579-ab213511'>ab213511</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T cells transfected with a full length human DYRK1A expression vector containing a mCherry tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a full length human DYRK1B expression vector containing a mCherry tag whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 86 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Low expression : liver and cerebellum.

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

All lanes:

Western blot - Anti-CD66b antibody [RM1323] (<a href='/en-us/products/primary-antibodies/cd66b-antibody-rm1323-ab325777'>ab325777</a>) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

human spleen tissue lysate at 20 µg

Lane 3:

Human cerebellum tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 95 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Negative control : SW1463 and HCC827.

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

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

All lanes:

Western blot - Anti-CD66b antibody [RM1323] (<a href='/en-us/products/primary-antibodies/cd66b-antibody-rm1323-ab325777'>ab325777</a>) at 1/1000 dilution

Lane 1:

SW1463 (human Large intestine Rectum epithelial) whole cell lysate at 20 µg

Lane 2:

HCC827 (human lung adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Untreated HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

Lane 4:

HL-60 treated with 1.25% Dimethyl sulfoxide (DMSO) for 5 days whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 95 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Low expression : Jurkat, A20, EL4

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 29540552).

The identity of the lower MW bands at approximately 60 kDa are unknown.

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

All lanes:

Western blot - Anti-IB-1 antibody [EPR30423-508] (<a href='/en-us/products/primary-antibodies/ib-1-antibody-epr30423-508-ab325525'>ab325525</a>) at 1/1000 dilution

Lane 1:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 40 µg

Lane 3:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 40 µg

Lane 4:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 40 µg

Lane 5:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 110 kDa,120 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Low expression : 293T(PMID : 10934049)

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) 1 : 10000 (124Kda).

All lanes:

Western blot - Anti-PTPN14 antibody [EPR30487-534] (<a href='/en-us/products/primary-antibodies/ptpn14-antibody-epr30487-534-ab326098'>ab326098</a>) at 1/1000 dilution

Lane 1:

DU 145 (human prostate carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 80 µg

Lane 2:

DU 145 transfected with siRNA specifically targeting PTPN14 whole cell lysate at 80 µg

Lane 3:

293T (human embryonic kidney epithelial cell) whole cell lysate at 80 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 135 kDa,124 kDa

false

Exposure time: 135s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Negative control : HeLa, HT-29, Jurkat.

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

All lanes:

Western blot - Anti-CD137 antibody [EPR30968-503] (<a href='/en-us/products/primary-antibodies/cd137-antibody-epr30968-503-ab326426'>ab326426</a>) at 1/1000 dilution

Lane 1:

HDLM-2 (human hodgkin lymphoma cell) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 32 kDa,40 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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-E. coli antibody [EPR28234-47] (<a href='/en-us/products/primary-antibodies/e-coli-antibody-epr28234-47-ab326069'>ab326069</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

RAW264.7 co-cultured with E. coli BL21(DE3) bacteria for 1.5 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 15-37 kDa,36 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

To minimize protein degradation, cell was 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 36 kDa is unknown.

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

All lanes:

Western blot - Anti-Spastin antibody [EPR29584-166] (<a href='/en-us/products/primary-antibodies/spastin-antibody-epr29584-166-ab325650'>ab325650</a>) at 1/1000 dilution

All lanes:

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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 50-60 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Performed under reducing conditions.

In Western blot, ab325650 was shown to bind specifically to Spastin. Target of interest was observed at 50-60 kDa in wild-type HEK-293 cell lysates (lane 1) with no signal observed at this size in SPAST knockout cell line (lane 2) (lane 2, knockout cell line ab267238 / knockout cell lysate ab258698).

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 36 kDa is unknown.

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

All lanes:

Western blot - Anti-Spastin antibody [EPR29584-166] (<a href='/en-us/products/primary-antibodies/spastin-antibody-epr29584-166-ab325650'>ab325650</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 (human embryonic kidney epithelial cell) fresh whole cell lysate at 20 µg

Lane 2:

SPAST knockout HEK-293 fresh whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 50-60 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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 bands beneath the target band (100 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-QRICH1 antibody [EPR30491-560] (<a href='/en-us/products/primary-antibodies/qrich1-antibody-epr30491-560-ab326365'>ab326365</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Low expression : MCF7, LoVo.

250 kDa full length band and 120-130 kDa cleavage fragment band were ovserved (PMID : 20471383).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

All lanes:

Western blot - Anti-Robo1 antibody [EPR29534-534] (<a href='/en-us/products/primary-antibodies/robo1-antibody-epr29534-534-ab326048'>ab326048</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

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

Lane 6:

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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 120-130 kDa,250 kDa,124 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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).

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

All lanes:

Western blot - Anti-ECE1 antibody [EPR30300-555] (ab326103) at 1/1000 dilution

Lane 1:

PC-3 (human prostate adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

PC-3 transfected with siRNA specifically targeting ECE1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 130 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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-Cyclin D1 (phospho T286) antibody [EPR30260-527] (<a href='/en-us/products/primary-antibodies/cyclin-d1-phospho-t286-antibody-epr30260-527-ab325926'>ab325926</a>) at 1/1000 dilution

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 10µM MG-132 for 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 33 kDa,36 kDa

false

Exposure time: 92s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Performed under reducing conditions.

In Western blot, aab325926 was shown to bind specifically to Cyclin D1 (phospho T286). Target of interest was observed at 33 kDa in A549 treated with 10µM MG-132 for 4 hours lysates (lane 2) with no signal observed at this size in CCND1 (Cyclin D1) knockout cell line (lanes 3-4) (lanes 3-4, knockout cell line ab286759).

The identity of the bands higher than 75kda are unknown.

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

All lanes:

Western blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (<a href='/en-us/products/primary-antibodies/cyclin-d1-phospho-t286-antibody-epr30260-527-ab325926'>ab325926</a>) at 1/1000 dilution

Lane 1:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate 20 µg (untreated membrane)

Lane 2:

A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (untreated membrane)

Lane 3:

Untreated CCND1 (Cyclin D1) knockout A549 whole cell lysate 20 µg (untreated membrane)

Lane 4:

CCND1 (Cyclin D1) knockout A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (untreated membrane)

Lane 5:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 6:

A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 7:

Untreated CCND1 (Cyclin D1) knockout A549 whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 8:

CCND1 (Cyclin D1) knockout A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 33 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Tyrosine Hydroxylase antibody [EP1532Y] - Neuronal Marker (<a href='/en-us/products/primary-antibodies/tyrosine-hydroxylase-antibody-ep1532y-neuronal-marker-ab137869'>ab137869</a>) at 0.1 µg/mL

All lanes:

Human adrenal gland lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

false

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Performed under reducing conditions.

In Western blot, ab325917 was shown to bind specifically to NFAT1. Target of interest was observed at 90 kDa and 110 KDa in wild-type Raji cell lysates (lane 1) with no signal observed at this size in NFAT1 knockout cell line (lane 2) (lane 2, knockout cell line ab280906 / knockout cell lysate ab282940).

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

All lanes:

Western blot - Anti-NFAT1 antibody [EPR30445-509] (<a href='/en-us/products/primary-antibodies/nfat1-antibody-epr30445-509-ab325917'>ab325917</a>) at 1/1000 dilution

Lane 1:

Wild-type Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate at 20 µg

Lane 2:

NFAT1 knockout Raji whole cell lysate at at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 90 kDa,110 kDa,36 kDa

false

Exposure time: 133s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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 bands beneath the target band (100 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-QRICH1 antibody [EPR30491-560] (<a href='/en-us/products/primary-antibodies/qrich1-antibody-epr30491-560-ab326365'>ab326365</a>) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) transfected with scrambled siRNA control fresh whole cell lysate at 20 µg

Lane 2:

A549 transfected with siRNA specifically targeting QRICH1 fresh whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Collaborator

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

The data was kindly provided by a collaborator.

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:

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 ATP13A2 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 150 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

ab322735 was shown to react with ATP13A2 in wild-type HEK-293 cells in Western blot with loss of signal observed in ATP13A2 knockout cell line ab274925. Wild-type HEK-293 and ATP13A2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab322735 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging. This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

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:

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

Lane 2:

Western blot - Human ATP13A2 knockout HEK-293 cell lysate (<a href='/en-us/products/cell-lysates/human-atp13a2-knockout-hek-293-cell-lysate-ab274983'>ab274983</a>) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 150 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

Exposure time : Lanes 1-2 : 26 seconds; Lanes 3-8 : 59 seconds

All lanes:

Western blot - Anti-SMC1A antibody [EPR30532-7] (<a href='/en-us/products/primary-antibodies/smc1a-antibody-epr30532-7-ab325922'>ab325922</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T transfected with siRNA specifically targeting SMC1A fresh whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 7:

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

Lane 8:

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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 160 kDa,124 kDa

false

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

Exposure time

Lane 1 to 2 : 10 seconds
Lane 3 to 4 : 30 seconds

We recommend not to boil the samples after lysis to get desired WB bands.

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

All lanes:

Western blot - Anti-Glucose Transporter GLUT1 antibody [EPR3915] (<a href='/en-us/products/primary-antibodies/glucose-transporter-glut1-antibody-epr3915-ab115730'>ab115730</a>) at 1/50000 dilution

Lane 1:

HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates unboiled at 20 µg

Lane 2:

HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates boiled at 20 µg

Lane 3:

3T3-L1 (Mouse embryonic fibroblast) whole cell lysates unboiled at 20 µg

Lane 4:

3T3-L1 (Mouse embryonic fibroblast) whole cell lysates boiled at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 40-60 kDa

false

• WB

Lab

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

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

Low expression : TT, A431.

The bands beneath the target band (33 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-SHOX2 antibody [EPR30169-39] (<a href='/en-us/products/primary-antibodies/shox2-antibody-epr30169-39-ab326071'>ab326071</a>) at 1/1000 dilution

Lane 1:

MIA PaCa-2 (human pancreas epithelial cell) transfected with scrambled siRNA control whole cell lysate at 50 µg

Lane 2:

MIA PaCa-2 transfected with siRNA specifically targeting SHOX2 whole cell lysate at 50 µg

Lane 3:

TT (human thyroid carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 4:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 33-38 kDa,36 kDa

false

Exposure time: 114s