All lanes : Anti-HTF9C/TRMT2A antibody [EPR16485-54] (ab205616) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TRMT2A knockout HeLa cell lysate
Lane 3 : MOLT-4 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab205616 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab205616 Anti-HTF9C/TRMT2A antibody [EPR16485-54] was shown to specifically react with HTF9C/TRMT2A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265160 (knockout cell lysate ab258253) was used.  Wild-type and HTF9C/TRMT2A knockout samples were subjected to SDS-PAGE.  ab205616 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling HTF9C/TRMT2A with ab205616 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500.

Cytoplasmic staining on human breast carcinoma tissue is observed.

Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody with secondary antibody, ab97051 at 1/500.

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

Anti-HTF9C/TRMT2A antibody [EPR16485-54] (ab205616) at 1/1000 dilution + Human fetal kidney at 20 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and diluting buffer was 5% NFDM /TBST

All lanes : Anti-HTF9C/TRMT2A antibody [EPR16485-54] (ab205616) at 1/5000 dilution

Lane 1 : MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate
Lane 2 : HEK293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking and diluting buffer was 5% NFDM /TBST

Exposure time Lane 1 and 2: 10 seconds
Lane 3: 1 minute

Anti-HTF9C/TRMT2A antibody [EPR16485-54] (ab205616) at 1/1000 dilution + HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

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

Developed using the ECL technique.

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking and diluting buffer was 5% NFDM /TBST

All lanes : Anti-HTF9C/TRMT2A antibody [EPR16485-54] (ab205616) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 69 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking and diluting buffer was 5% NFDM /TBST

HTF9C/TRMT2A was immunoprecipitated from 1mg of HEK293 (Human epithelial cell line from embryonic kidney) whole cell extract with ab205616 at 1/100.

Western blot was performed from the immunoprecipitate using ab205616 at 1/5000 followed by VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/10000.

Lane 1: HEK293 whole cell extract at 10ug

Lane 2: HEK293 whole cell extract immunoprecipitated using ab205616

Lane 3: HEK293 whole cell extract immunoprecipitated using ab172730 (Rabbit monoclonal IgG)

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