N-Cadherin Antibody (13A9) - BSA Free

Western Blot Protocol
1. Perform SDS-PAGE (4-12% MOPS) on samples to be analyzed, loading 25 ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.
3. Rinse membrane with dH2O and then stain the blot using Ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% NFDM + 1% BSA in TBS + Tween, 1 hour at RT.
6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
7. Dilute the anti-N Cadherin primary antibody (NBP1-48309) in blocking buffer and incubate 1 hour at room temperature.
8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
9. Apply the diluted mouse-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions (Pierce ECL).
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.


Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.