Anti-CNPase antibody [11-5B] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CNPase antibody [11-5B] - BSA and Azide free (AB237961)

IHC image of CNPase staining in human cerebral cortex formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab6319, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CNPase antibody [11-5B] - BSA and Azide free (AB237961)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab6319).

ab6319 staining CNPase in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab6319 at 1μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• WB

Supplier Data

Western blot - Anti-CNPase antibody [11-5B] - BSA and Azide free (AB237961)

Lanes 1 - 3 : Merged signal (red and green). Green - ab6319 observed at 48 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab6319 was shown to recognize CNPase in wild-type HAP1 cells as signal was lost at the expected MW in CNPase knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CNPase knockout samples were subjected to SDS-PAGE. ab6319 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

All lanes:

Western blot - Anti-CNPase antibody [11-5B] (<a href='/en-us/products/primary-antibodies/cnpase-antibody-11-5b-ab6319'>ab6319</a>) at 5 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

CNPase knockout HAP1 whole cell lysate at 40 µg

Lane 3:

Human Brain whole cell lysate at 20 µg

Predicted band size: 48 kDa

false

• WB

Lab

Western blot - Anti-CNPase antibody [11-5B] - BSA and Azide free (AB237961)

This data was developed using the same antibody clone in a different buffer formulation (ab6319).

Lanes 1- 2 : Merged signal (red and green). Green - ab6319 observed at 48 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

ab6319 was shown to react with CNPase in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264949 (knockout cell lysate ab256877) was used. Wild-type HeLa and CNP knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab6319 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 5 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CNPase antibody [11-5B] (<a href='/en-us/products/primary-antibodies/cnpase-antibody-11-5b-ab6319'>ab6319</a>) at 5 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 1:

Western blot - Human CNP (CNPase) knockout HeLa cell pellet (<a href='/en-us/products/cell-lysates/human-cnp-cnpase-knockout-hela-cell-pellet-ab278945'>ab278945</a>)

Lane 1:

Western blot - Human CNP (CNPase) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-cnp-cnpase-knockout-hela-cell-lysate-ab256877'>ab256877</a>)

Lane 2:

CNP knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CNP (CNPase) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cnp-cnpase-knockout-hela-cell-line-ab264949'>ab264949</a>)

Predicted band size: 48 kDa

Observed band size: 48 kDa

false

• WB

Supplier Data

Western blot - Anti-CNPase antibody [11-5B] - BSA and Azide free (AB237961)

This antibody was raised against full length native CNPase and is predicted to recognize both isoforms. The predicted molecular weights of isoforms CNPI and CNPII are 45- and 48-kDa respectively.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

All lanes:

Western blot - Anti-CNPase antibody [11-5B] (<a href='/en-us/products/primary-antibodies/cnpase-antibody-11-5b-ab6319'>ab6319</a>) at 1/100 dilution

Lane 1:

Human spinal cord tissue lysate - total protein (<a href='/en-us/products/unavailable/human-spinal-cord-tissue-lysate-total-protein-ab29188'>ab29188</a>) at 20 µg

Lane 2:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 20 µg

Lane 3:

Spinal Cord (Mouse) Tissue Lysate at 20 µg

Lane 4:

Brain (Mouse) Tissue Lysate at 20 µg

Lane 5:

Spinal Cord (Rat) Tissue Lysate at 20 µg

Lane 6:

Brain (Rat) Tissue Lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 48 kDa

true

Exposure time: 1min