Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Protocol
Select your Protocol
Western Blotting
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Western Blotting Protocol
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween
®
20 at 4°C with gentle shaking, overnight.
NOTE
: Please refer to primary antibody product webpage for recommended antibody dilution.
A. Solutions and Reagents
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit:
#12957
Western Blotting Application Solutions Kit
NOTE
: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
20X Phosphate Buffered Saline (PBS)
: (
#9808
) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH
2
O, mix.
10X Tris Buffered Saline (TBS)
: (
#12498
) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH
2
O, mix.
1X SDS Sample Buffer
: Blue Loading Pack (
#7722
) or Red Loading Pack (
#7723
) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH
2
O.
10X Tris-Glycine SDS Running Buffer
: (
#4050
) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH
2
O, mix.
10X Tris-Glycine Transfer Buffer
: (
#12539
) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH
2
O, mix.
10X Tris Buffered Saline with Tween
®
20 (TBST)
: (
#9997
) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH
2
O, mix.
Nonfat Dry Milk
: (
#9999
).
Blocking Buffer
: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
Wash Buffer
: (
#9997
) 1X TBST.
Bovine Serum Albumin (BSA)
: (
#9998
).
Primary Antibody Dilution Buffer
: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
Biotinylated Protein Ladder Detection Pack
: (
#7727
).
Blue Prestained Protein Marker, Broad Range (11-250 kDa)
: (
#59329
).
Blotting Membrane and Paper
: (
#12369
) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
Secondary Antibody Conjugated to HRP
: Anti-rabbit IgG, HRP-linked Antibody (
#7074
).
Detection Reagent
: SignalFire™ ECL Reagent (
#6883
).
B. Protein Blotting
A general protocol for sample preparation.
Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells with 1X PBS; aspirate.
Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
Microcentrifuge for 5 min.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE
: Loading of prestained molecular weight markers (
#59329
, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (
#7727
, 10 µl/lane) to determine molecular weights are recommended.
Electrotransfer to nitrocellulose membrane (
#12369
).
C. Membrane Blocking and Antibody Incubations
NOTE
: Volumes are for 10 cm x 10 cm (100 cm
2
) of membrane; for different sized membranes, adjust volumes accordingly.
I. Membrane Blocking
(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.
II. Primary Antibody Incubation
Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
Wash three times for 5 min each with 15 ml of TBST.
Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (
#7074
at 1:2000) and anti-biotin, HRP-linked Antibody (
#7075
at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.
Proceed with detection (Section D).
D. Detection of Proteins
Directions for Use:
Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
Prepare 1X SignalFire™ ECL Reagent (
#6883
) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Specificity / Sensitivity
GIT2 Antibody recognizes endogenous levels of total GIT2 protein.
Human, Monkey
Source / Purification
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human GIT2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Background
G protein-coupled receptor (GPCR) kinase interacting proteins 1 and 2 (GIT1 and GIT2) are highly conserved, ubiquitous scaffold proteins involved in localized signaling to help regulate focal contact assembly and cytoskeletal dynamics. GIT proteins contain multiple interaction domains that allow interaction with small GTPases (including ARF, Rac, and cdc42), kinases (such as PAK and MEK), the Rho family GEF Pix, and the focal adhesion protein paxillin (reviewed in 1). GIT1 and GIT2 share many of the same properties, but with at least ten distinct, tissue-specific splice variants. GIT2 has been shown to play an important role inhibiting focal adhesion turnover and membrane protrusion (2,3). Focal adhesion localization and paxillin binding of GIT2 is regulated through phosphorylation at one or more tyrosine sites (Tyr286, Tyr392, Tyr592) by FAK and/or Src (4,5,reviewed in 6). Once at the focal adhesion, GIT2 is thought to play a key role in cell polarity and migration, making it a protein of interest in the investigation of oncogenic signaling pathways (3,5,7).
Hoefen, R.J. and Berk, B.C. (2006)
J Cell Sci
119, 1469-75.
Premont, R.T. et al. (2000)
J Biol Chem
275, 22373-80.
Frank, S.R. et al. (2006)
EMBO J
25, 1848-59.
Brown, M.C. et al. (2005)
Mol Biol Cell
16, 4316-28.
Yu, J.A. et al. (2009)
Mol Biol Cell
20, 4706-19.
Yu, J.A. et al. (2010)
Cell Adh Migr
4, 342-7.
Mazaki, Y. et al. (2006)
Nat Immunol
7, 724-31.
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