RAB35 Rabbit pAb

Blank control:A549. Primary Antibody (green line): Rabbit Anti-RAB35 antibody (AP94111) Dilution: 1 µg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-PE Dilution:0.2 µg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 20% PBST for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Paraformaldehyde-fixed, paraffin embedded (Human breast cancer); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (RAB35) Polyclonal Antibody, Unconjugated (AP94111) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

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Product Information
Application Applications Legend: E=ELISA WB=Western Blotting IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin) IP=Immunoprecipitation IF=Immunofluorescence IC=Immunochemistry ICC=Immunocytochemistry FC=Flow Cytometry DB=Dot Blot	IHC-P, IHC-F, IF
Reactivity	Human
Host	Rabbit
Clonality	Polyclonal
Calculated MW	22 KDa
Physical State	Liquid
Immunogen	KLH conjugated synthetic peptide derived from human RAB35
Epitope Specificity	131-201/201
Isotype	IgG
Purity	affinity purified by Protein A
Buffer	0.01M TBS (pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.
SUBCELLULAR LOCATION	Cell membrane; Lipid-anchor; Cytoplasmic side (Potential). Membrane, clathrin-coated pit. Cytoplasmic vesicle, clathrin-coated vesicle. Endosome. Melanosome.
SIMILARITY	Belongs to the small GTPase superfamily. Rab family.
SUBUNIT	Interacts with DENND1A and DENND1B in a nucleotide-dependent manner. The low binding to DENND1C is nucleotide-independent. All 3 DENND1 act as GEFs for RAB35 and thus activate the protein.
Post-translational modifications	AMPylation at Tyr-77 by L.pneumophila DrrA occurs in the switch 2 region and leads to moderate inactivation of the GTPase activity. It appears to prolong the lifetime of the GTP state of RAB1B by restricting access of GTPase effectors to switch 2 and blocking effector-stimulated GTP hydrolysis, thereby rendering RAB35 constitutively active.Phosphocholinated by L.pneumophila AnkX. Both GDP-bound and GTP-bound forms can be phosphocholinated. Phosphocholination inhibits the GEF activity of DENND1A.
Important Note	This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications.
Background Descriptions	In the process of endocytosis, essential rate-limiting regulator of a fast recycling pathway back to the plasma membrane. During cytokinesis, required for the postfurrowing terminal steps, namely for intercellular bridge stability and abscission, possibly by controlling phosphatidylinositol 4,5-bis phosphate (PIP2) and SEPT2 localization at the intercellular bridge.

Additional Information
Dilution	IHC-P=1:100-500,IHC-F=1:100-500,IF=1:100-500,Flow-Cyt=0.2ug/test
Format	0.01M TBS(pH7.4) with 1% BSA, 0.09% (W/V) sodium azide and 50% Glyce
Storage	Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C.

Research Areas

For Research Use Only. Not For Use In Diagnostic Procedures.

Application Protocols

Provided below are standard protocols that you may find useful for product applications.

Western Blot Protocol

Blocking Peptides Protocol

Dot Blot Protocol

Immunohistochemistry Protocol

Immunofluorescence Protocol

Immunoprecipitation Protocol

Flow Cytomety Protocol

Cell Culture Protocol