CDC42EP1 Antibody

CDC42EP1, also known as CDC42 Effector Protein 1, consists of a 40.2 kDa and 39.7 kDa isoform, and is involved in regulatory processes within the cell as a member of the Rho GTPase family. Most commonly found in bone marrow, the CDC42EP1 proteins been shown to help facilitate actin cytoskeleton restructuring and cell shape regulation. The protein is being studied for its involvement in osteonecrosis. The protein has been linked to the RhoA and Rho GTPases pathways where it interacts with MAPKAP1, RPTOR, RHOQ, CDC42, RBPMS, KRTAP4-12, PLSCR1.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Images	Ratings	Applications	Species	Date	Details
Enlarge	5 reviewed by: Stephen Terry	ICC	Human	10/25/2018	View Summary Application Immunocytochemistry Sample Tested hela cell Species Human Lot A96959 Comments Comments Cells fixed with 4% PFA for 20 minutes room temperature. Fixed cells incubated with with 0.5% triton x100 in 1xPBS for 5 minutes room. Washed twice in blocking buffer (1% BSA 1xPBS, 20mM glycine). Washed once with blocking buffer left for 1hour room temperature. incubated with Anti Cdc42EP1 antibody in blocking buffer 1:200 for 20 hours at 4 degrees. Washed 3 x 5 minutes in blocking buffer Incubated with Anti rabbit-Alexa 594 conjugated antibody (Jackson immunoscience) for 2 hours room temperature. Washed 3 x 5 minutes in blocking buffer Washed 1 x 5 minutes in PBS Imaged with 60X oil objective on Nikon Eclipse Ti Inverted Fast confocal - Equipped with a Yokogawa CSU-X1 spinning disk unit and okogawa CSU-1 disk head and Andor Neo sCMOS camera.
Enlarge	5 reviewed by: Stephen Terry	IP	Human	08/20/2018	View Summary Application Immunoprecipitation Sample Tested hela cell Species Human Lot A96959 Comments Comments Vortex tube of mag protein G beads (NEB) Take 30 μl put in tube Add 1 ml of 1XPBS to wash Put on mag rack discard PBS Suspend beads in 60 μl of 1XPBS Add between 2mg of primary IP antibody to beads Put on rotating wheel at 4°C for 20h Lysis of cells Put all microfuge tubes that you are going to use on ice Cool centrifuge to 4°C Take HeLa cells grown on 10cm dishes to about 70% confluence Aspirate media wash twice with 10ml cold 1xPBS Add 1 ml of lysis buffer + 1xphosphatase + 1xprotease inhibtors (Roche) to each 10cm plate Scrape cells off plate Syringe 7-8 times with 25 G needle Transfer to microfuge tube on ice pre clear lysate by adding 60 μl of mag protein G beads (NEB) to each 1 ml of lysate Leave on ice for 10 min inverting several times. Spin tubes in cool centrifuge max speed 13K rpm for 5 min Put tubes in mag rack pipette off supernatant Save 50 μl of one of the samples to use as an input lane add 50 μl of 3X sample buffer +0.15M DTT to this boil for 5 min store -20°C save as input -load 20ul of this on gel Add 1ml of pre cleared lysate to each of the protein G –Antibody conjugates prepared day before Put on rotating wheel in cold room for 2h Put tubes on mag rack Wash once with 1ml cold 0.5% triton x100 PBS Put beads on rack remove liquid Wash 3X more with 1ml cold 1XPBS Suspend beads in 80 μl of 3X of 3X sample buffer +0.3MDTT boil 10 min Put on mag rack Transfer liquid to new tube Load 15 μl on gel along with 20 μl of inputs Gel run out on 10% SDS PAGE gel in MOPS buffer Transfered 100V 2hours 4 degrees nitrocellulose membrane blocked 5% milk PBST 1 hour incubated overnight with Cdc42EP1 antibody NBP1-88379 1:2000 in 5% milk PBST Washed 3X PBST Incubated with secondary anti rabbit HRP con light chain specific antibody 1:5000 (Jackson immunoresearch 211-032-171) in for 1hour 5% milk PBST Washed 3X PBST washed once 1X PBS ECL prime kit GE healthcare used for detection of chemilumincence
Enlarge	5 reviewed by: Stephen Terry	WB	Human	08/03/2018	View Summary Application Western Blot Sample Tested Hela whole cell lysate Species Human Lot A96959 Comments Comments For siRNA blot two different siRNAs were transfected at 40nM, targeting cdc42ep1 or non targeting control siRNA cells were lysed after 72 hours post tranfection Protein lysates were lysed in 1.5X Lammeli sample buffer boiled at 100 degrees for 5 minutes 10ug of protein loaded on 10% SDS PAGE gel run in MOPS buffer I used the antibody at 1:2000 in 5% milk TBST incubation overnight 4 degrees antibody gives a band at -55kD that reduces in RNAi Membranes were reprobed with GAPDH antibody as a loading control

Array NBP1-88379

• CDC42EP1 Antibodies
• CDC42EP1 Lysates
• CDC42EP1 Proteins