Anti-alpha Tubulin antibody [EP1332Y] - Loading Control

26 product images

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling alpha Tubulin with ab52866 at 1/500 dilution. The cells were permeabilised with 0.1% Triton X-100. Anti-rabbit Alexa Fluor® 488 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/400 dilution was used as the secondary antibody (green). The confocal image shows microtubules staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 and anti-mouse AlexaFluor® 594 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 1/500 dilution (red).
  The negative controls are as follows:
  1. ab52866 at 1/500 dilution followed by anti-mouse AlexaFluor® 594 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 1/500 dilution.
  2. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by anti-rabbit Alexa Fluor® 488 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/400 dilution.

• 

  Image from Hotte K. et al Biol Open. 2017 Jun 15;6(6):723-731. doi: 10.1242/bio.022335.

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866)

  AJAP1 co-localizes with microtubules in HUVECs

  The association of AJAP1 with microtubules in HUVECs is lost upon microtubule destruction. Treatment with 12.5 μM nocodazole for 24 h shows destruction of the microtubule network and loss of AJAP1 tubular localization. For a negative control, HUVECs are treated with DMSO for 24 h. Cell nuclei were counterstained with DAPI (cyan). Microscope: Zeiss LSM 780; objective lens: 63×/1.40 oil; scale bar: 25 μm.

  Incubated overnight at 4°C with ab52866.

  (From Figure 3E of Hotte et al)

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

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

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/10000 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) Whole cell lysates at 15 µg

  Lane 2: PC-12 (Rat adrenal gland heochromocytoma) whole cell lysates at 15 µg

  Lane 3: NIH/3T3( Mouse embryonic fibroblast)whole cell lysates at 15 µg

  Secondary

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

  Predicted band size: 50 kDa

  Observed band size: 52 kDa

  Exposure time: 1s

• 

  This image is courtesy of an anonymous customer review.

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866)

  ab52866 staining alpha Tubulin in 293 Human embryonic kidney cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 10% serum for 2 hours at 23°C. Samples were incubated with primary antibody (1/200 in 0.5% saponin) for 2 hours at 23°C. An Alexa Fluor^®555-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody. Nuclei were counterstained with DAPI.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-alpha Tubulin antibody

  Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling alpha Tubulin with ab52866 at followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human stomach.The section was incubated with Anti-DLL3 antibody [EPR22592-18] ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

• 

  Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Flow Cytometry (Intracellular) staining using rabbit Anti-alpha Tubulin antibody

  Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (Human hepatocellular carcinoma epithelial cell)cells labelling alpha Tubulin with ab52866 at 1/2000 dilution (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730)isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
  

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/5000 dilution

  Lane 1: Mouse brain lysates at 10 µg

  Lane 2: C6 (Rat glial tumor cell line) whole cell lysates at 10 µg

  Lane 3: RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysates at 10 µg

  Lane 4: PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysates at 10 µg

  Lane 5: NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysates at 10 µg

  Secondary

  All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

  Predicted band size: 50 kDa

• 

  Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Flow Cytometry (Intracellular) staining using rabbit Anti-alpha Tubulin antibody

  Intracellular Flow Cytometry analysis of 2% paraformaldehyde fixed HepG2 (human liver hepatocellular carcinoma cell line) cells labeling alpha Tubulin with ab52866 at 1/130 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).
  

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/20000 dilution

  Lane 1: HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Lane 2: HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

  Lane 3: Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

  Lane 4: HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

  Predicted band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining of Rat brain lysates using rabbit Anti-alpha Tubulin antibody

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/50000 dilution

  All lanes: Rat brain lysates at 10 µg

  Secondary

  All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

  Predicted band size: 50 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866)

  Immunohistochemistry analysis of paraffin-embedded Rat kidney tissue labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on Rat kidney tubule and weak on glomerulus shown. Secondary antibody Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) used at a 1/500 dilution. Counter stained with Hematoxylin.
  

  Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866)

  Immunohistochemistry analysis of paraffin-embedded Mouse kidney tissue labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on Mouse kidney tubule shown. Secondary antibody Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) used at a 1/500 dilution. Counter stained with Hematoxylin.
  

  Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-alpha Tubulin antibody

  Immunohistochemistry analysis of paraffin-embedded Human breast cancer labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on cancer cells shown. Secondary antibody Goat Anti-Rabbit IgG H&L (HRP) ab97051 Goat Anti-Rabbit IgG H&L (HRP) used at a 1/500 dilution. Counter stained with Hematoxylin.
  

  Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining of Human fetal kidney lysates using rabbit Anti-alpha Tubulin antibody

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/50000 dilution

  All lanes: Human fetal kidney lysates at 10 µg

  Secondary

  All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

  Predicted band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab52866 overnight at 4°C. Antibody binding was detected using Anti-Rabbit Alexa Fluor® 790 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) ab175781) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/1000 dilution

  Lane 1: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

  Lane 2: HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

  Lane 3: HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 20 µg

  Lane 4: Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 20 µg

  Lane 5: NIH/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg

  Lane 6: PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) ab175781) at 1/10000 dilution

  Predicted band size: 50 kDa

  Observed band size: 52 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining of Pig skeletal muscle lysates using rabbit Anti-alpha Tubulin antibody

  All lanes: Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866) at 1/5000 dilution

  All lanes: Pig skeletal muscle lysates at 20 µg

  Secondary

  All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

  Predicted band size: 50 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-alpha Tubulin antibody

  Immunohistochemistry analysis of paraffin-embedded Pig kidney tissue labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on Pig kidney tubule and weak on glomerulus shown. Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) used at a 1/100 dilution. Counter stained with Hematoxylin.
  

  Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100 dilution.

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  Western blot: anti-ACK antibody staining at 1/1000 dilution, shown in green; Rabbit anti alpha Tubulin (ab52866) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 118 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in TNK2 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

  All lanes: Western blot at 1/1000 dilution

  Lane 1: Wild-type HCT 116 at 20 µg

  Lane 2: Western blot - Human TNK2 knockout HCT116 cell line (ab301274) at 20 µg

  Lane 3: HeLa at 20 µg

  Lane 4: PC-3

  Secondary

  All lanes: Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

  Performed under reducing conditions.

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  Western blot: Mouse Monoclonal[2D2] to ARID1B Anti-ARID1B antibody [2D2] ab57461 staining at 1/1000 dilution, shown in green; Rabbit anti alpha Tubulin (ab52866) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 200 kDa in Wild-type HCT 116 Nuclear cell lysates with no signal observed at this size in ARID1B knockout HCT 116 Nuclear cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-ARID1B antibody [2D2] (Anti-ARID1B antibody [2D2] ab57461) at 1/1000 dilution

  Lane 1: Wild-type HCT 116 Nuclear at 20 µg

  Lane 2: Western blot - Human ARID1B knockout HCT116 cell line (Human ARID1B knockout HCT116 cell line ab287219) at 20 µg

  Lane 3: RKO at 20 µg

  Lane 4: PC-3 Membrane at 20 µg

  Secondary

  All lanes: Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 244 kDa

  Observed band size: 200 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  Western blot: TIGAR Antibody (E-2): sc-166290 absc-166290 staining at 1/1000 dilution, shown in green; Rabbit anti alpha Tubulin (ab52866) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 30 kDa in Wild-type A549 cell lysates with no signal observed at this size in TIGAR knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

  All lanes: Western blot at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: TIGAR knockout A549 at 20 µg

  Lane 3: HepG2 at 20 µg

  Lane 4: SK-OV-3 at 20 µg

  Secondary

  All lanes: Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

  Performed under reducing conditions.

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  False colour image of Western blot: Anti-PVRIG/CD112R antibody staining at 1 ug/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red.In Western blot, Anti-PVRIG/CD112R antibody ab168153 was found to be non-specific.
  
  A band was observed at 38 kDa in wild-type Jurkat cell lysates with no change observed in the PVRIG knockout cell line Human PVRIG knockout Jurkat cell line ab274900 (knockout cell lysate Human PVRIG knockout Jurkat cell lysate ab274958). To generate this image, wild-type and PVRIG knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane.
  
  Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.

  All lanes: Western blot - Anti-PVRIG/CD112R antibody (Anti-PVRIG/CD112R antibody ab168153) at 1 µg/mL

  Lane 1: Wild-type Jurkat cell lysate at 20 µg

  Lane 2: PVRIG knockout Jurkat cell lysate at 20 µg

  Lane 3: Daudi cell lysate at 20 µg

  Lane 4: Daudi cell lysate at 10 µg

  Lane 5: HeLa cell lysate at 20 µg

  Secondary

  Lanes 1 - 5: Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution

  Lanes 1 - 5: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 34 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866)

  Immunohistochemical analysis of formalin fixed paraffin embedded human kidney staining alpha Tubulin with ab52866 at a concentration of 0.01 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  ab52866 anti- alpha Tubulin [EP1332Y] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  Western blot: Anti-MGMT antibody [MT3.1] (Anti-MGMT antibody [MT3.1] ab39253) staining at 1/500 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-MGMT antibody [MT3.1] ab39253 was shown to bind specifically to MGMT. A band was observed at 22 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MGMT knockout cell line Human MGMT knockout HCT116 cell line ab286541. The band observed in the knockout lysate lane below 22 kDa is likely to represent a truncated form of MGMT. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MGMT knockout HCT 116 cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-MGMT antibody [MT3.1] (Anti-MGMT antibody [MT3.1] ab39253) at 1/500 dilution

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: Western blot - Human MGMT knockout HCT116 cell line (Human MGMT knockout HCT116 cell line ab286541)

  Lane 2: MGMT knockout HCT 116 cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 2: Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 22 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  Western blot: Anti-SEMA4D antibody [30/CD100] (Anti-Semaphorin 4D/CD100 antibody [30/CD100] ab307685) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Semaphorin 4D/CD100 antibody [30/CD100] ab307685 was shown to bind specifically to SEMA4D. A band was observed at 90-120 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in SEMA4D knockout cell line. To generate this image, wild-type and SEMA4D knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (Anti-Semaphorin 4D/CD100 antibody [30/CD100] ab307685) at 1/1000 dilution

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: Western blot - Human SEMA4D knockout HCT116 cell line (Human SEMA4D knockout HCT116 cell line ab289144)

  Lane 2: SEMA4D knockout HCT 116 cell lysate at 20 µg

  Lane 3: HDLM-2 UNBOILED cell lysate at 20 µg

  Lane 4: PC-3 UNBOILED cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 4: Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 90-120 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  False colour image of Western blot: Anti-MAGEA4 antibody [OTI1F9] staining at 1/4000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-MAGEA4 antibody [OTI1F9] ab139297 was shown to bind specifically to MAGEA4. A band was observed at 40 kDa in wild-type A431 cell lysates with no signal observed at this size in MAGEA4 knockout cell line Human MAGEA4 knockout A-431 cell line ab273857 (knockout cell lysate ab273811). To generate this image, wild-type and MAGEA4 knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-MAGEA4 antibody [OTI1F9] (Anti-MAGEA4 antibody [OTI1F9] ab139297) at 1/4000 dilution

  Lane 1: Wild-type A431 cell lysate at 20 µg

  Lane 2: Western blot - Human MAGEA4 knockout A-431 cell lysate (ab273811) at 20 µg

  Lane 3: U-2 OS cell lysate at 20 µg

  Lane 4: SH-SY5Y cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 35 kDa

  Observed band size: 40 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [EP1332Y] - Loading Control (ab52866)

  Alpha Tubulin Western blot staining using rabbit Anti-alpha Tubulin antibody

  Anti-THY1 antibody [7E1B11] (Anti-CD90 / Thy1 antibody [7E1B11] ab181469) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-CD90 / Thy1 antibody [7E1B11] ab181469 was shown to bind specifically to THY1. A band was observed at 25-37 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in THY1 knockout cell line Human THY1 (CD90) knockout U-2 OS cell line ab262490 (knockout cell lysate Human THY1 (CD90) knockout U-2 OS cell lysate ab263925). To generate this image, wild-type and THY1 knockout U-2 OS cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-CD90 / Thy1 antibody [7E1B11] (Anti-CD90 / Thy1 antibody [7E1B11] ab181469) at 1/1000 dilution

  Lanes 1 - 5: Western blot at 20 µg

  Lane 2: Western blot - Human THY1 (CD90) knockout U-2 OS cell lysate (Human THY1 (CD90) knockout U-2 OS cell lysate ab263925)

  Lane 2: Western blot - Human THY1 (CD90) knockout U-2 OS cell line (Human THY1 (CD90) knockout U-2 OS cell line ab262490)

  Secondary

  All lanes: Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 17 kDa