VEGFR2/KDR/Flk-1 Antibody - BSA Free

VEGFR2/KDR/Flk-1 Antibody:
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.

VEGFR2/KDR/Flk-1 Antibody:
Western Blot Protocol

1. Perform SDS-PAGE (3-8%) on samples to be analyzed, loading 50 ug of total protein per lane.

2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.

3. 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% non-fat dry milk + 0.5% BSA in TBS for 1 hour.

6. Dilute the rabbit anti-VEGFR2 primary antibody (NB 100-529) in blocking buffer and incubate 2 hours at room temperature.

7. Wash the membrane in water for 5 minutes and apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) and incubate 1 hour at room temperature.

8. Wash the blot in TBS containing 0.05-0.1% Tween-20 for 10-20 minutes.

9. Wash the blot in type I water for an additional 10-20 minutes (this step can be repeated as required to reduce background).

10. Apply the detection reagent of choice in accordance with the manufacturer's instructions (Amersham's ECL is the standard reagent used at Novus Biologicals).

Note: Tween-20 can be added to the blocking buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.