Cat. # | Size | Qty. | Price |
---|---|---|---|
3305T | 20 µl |
|
|
3305S | 100 µl |
|
REACTIVITY | H M R Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 94 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
Immunofluorescence (Immunocytochemistry) | 1:200 - 1:800 |
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.
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.
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.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
Proceed to one of the following specific set of steps.
NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2008
revised October 2021
Protocol Id: 409
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
Human, Mouse, Rat, Monkey
Bovine, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of human GCN5L2.
General Control of Amino Acid Synthesis Yeast Homolog Like 2 (GCN5L2) is a transcription adaptor protein and a histone acetyltransferase (HAT) that functions as the catalytic subunit of the STAGA and TFTC transcription coactivator complexes (1). GCN5L2 is 73% homologous to the p300/CBP-associated factor PCAF, another HAT protein found in similar complexes (2). Free GCN5L2 acetylates histone H3 on Lys14; however, when part of coactivator complexes, GCN5L2 acetylates histone H3 at Lys9, 14, 18, and 23, and to a smaller extent histones H4 and H2B (3). Histone acetylation contributes to gene activation by modulating chromatin structure and recruiting additional coactivator proteins that contain acetyl-lysine binding bromodomains (4). GCN5L2 also acetylates non-histone proteins such as transcription activators (TAT, c-Myb) (5,6), transcription co-activators (PGC1-α) (7), and nuclear receptors (Steroidogenic Factor 1) (8). Acetylation of these proteins regulates their nuclear localization, protein stability, DNA binding, and co-activator association (5-8). GCN5L2 is recruited to gene promoters during transactivation through interactions with multiple transcription activator proteins such as Myc, E2F, p53, and BRCA1 (9-12). The STAGA and TFTC complexes also interact with SAP130 and DDB1, two structurally related proteins involved in RNA splicing and DNA repair, suggesting roles for GCN5L2 in processes other than transcription activation (13).
Explore pathways related to this product.
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