The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
1/2000 - 1/10000. Detects a band of approximately 189 kDa (predicted molecular weight: 189 kDa).
Use at 2-5 µg/mg of lysate.
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Binds to activated CDC42 but does not stimulate its GTPase activity. It associates with calmodulin. Could serve as an assembly scaffold for the organization of a multimolecular complex that would interface incoming signals to the reorganization of the actin cytoskeleton at the plasma membrane. May promote neurite outgrowth.
Expressed in the placenta, lung, and kidney. A lower level expression is seen in the heart, liver, skeletal muscle and pancreas.
Regions C1 and C2 can either interact with nucleotide-free CDC42, or interact together, depending on the phosphorylation state of Ser-1443. When Ser-1443 is not phosphorylated, C1 and C2 interact, which prevents binding of nucleotide-free CDC42 and promotes binding of GTP-bound CDC42. Phosphorylation of Ser-1443 prevents interaction between C1 and C2, which opens the structure of the C-terminus and allows binding and sequestration of nucleotide-free CDC42 on both C1 and C2.
Phosphorylation of Ser-1443 by PKC prevents interaction between C1 and C2, allowing binding of nucleotide-free CDC42. Ser-1443 phosphorylation enhances the ability to promote neurite outgrowth.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling IQGAP1 with ab86064 at 1/1000 (0.2µg/ml) and 1/200 (1µg/ml). Detection: DAB and a DyLight® 594-conjugated goat anti-rabbit IgG (H+L) (1/100).
Western blot - IQGAP1 antibody (ab86064)
All lanes : Anti-IQGAP1 antibody (ab86064) at 0.04 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg
Predicted band size : 189 kDa Observed band size : 189 kDa Additional bands at : 238 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 10 seconds
Immunoprecipitation - IQGAP1 antibody (ab86064)
Detection of IQGAP1 by Western Blot of Immunprecipitate.
ab86064 at 0.4µg/ml staining IQGAP1 in HeLa whole cell lysate immunoprecipitated using ab86064 at 3µg/mg lysate (1 mg/IP; 20% of IP loaded/lane). Detection: Chemiluminescence with exposure time of 10 seconds.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IQGAP1 antibody (ab86064)Image courtesy of an anonymous Abreview.
ab86064 staining IQGAP1 in human pacreatic islets by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed in paraformaldehyde and a heat mediated antigen retrieval step was performed using citrate. Samples were then blocked with 5% serum for 1 hour at 20°C and then incubated with ab86064 at a 1/100 dilution for 8 hours at 4°C. The secondary used was a biotin conjugated goat anti-rabbit polyclonal used at a 1/1000 dilution.
ab86064 stained SKNSH cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab86064 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in Methanol fixed (100%, 5min) SKNSH cells at 1ug/ml.