Genetic Strategies: Western Blot: CDC42EP1 Antibody [NBP1-88379] - HeLa whole cell lysate. Lanes 1 and 4: HeLa cells transfected with control non-targeting siRNA. Lanes 2 and 3: HeLa cells transfected with two ...read more
Immunocytochemistry/ Immunofluorescence: CDC42EP1 Antibody [NBP1-88379] - Confocal Imunofluorescent images of HeLa cells fixed with PFA and stained with Cdc42EP1 antibody at 1:200 (magenta), and phalloidin-Alexa 488 ...read more
Immunohistochemistry-Paraffin: CDC42EP1 Antibody [NBP1-88379] - Staining of human stomach shows strong cytoplasmic positivity in glandular cells.
Immunocytochemistry/ Immunofluorescence: CDC42EP1 Antibody [NBP1-88379] - Staining of human cell line A-431 shows localization to plasma membrane & actin filaments. Antibody staining is shown in green.
Immunoprecipitation: CDC42EP1 Antibody [NBP1-88379] - Lanes 1 and 5: Marker. Lanes 2-4 HeLa cell input controls. Lane 6: IP using 2 mg of Control Rabbit IgG cell lysate. Lane 8: IP using 2 mg Cdc42EP1 cell lysate and ...read more
This antibody was developed against Recombinant Protein corresponding to amino acids: SGFCTISRLPRSEKPHDRDRDGSFPSEPGLRRSDSLLSFRLDLDLGPSLLSELLGVMSLPEAPAAETPAPAANPPAPTANPTGPAANPPATTANPPAPAANPSAPAATPTGPAANPPAPAASSTPHGHCPNGV
Immunogen affinity purified
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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
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
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