主页学习和支持蛋白质结构域和相互作用BROMO 蛋白质结构域BROMO 蛋白质结构域

Gcn5p 的 Bromodomain 结合乙酰化赖氨酸（红色）。

结构域结合和功能

从酵母到人的 100 多种蛋白质中发现，大约 110 个氨基酸的溴化域可以与乙酰化赖氨酸残基结合。乙酰化 N 端和 C 端组蛋白赖氨酸尾部残基，以及赖氨酸甲基化和丝氨酸/苏氨酸磷酸化，是翻译后修饰的重要形式，有助于将组蛋白与染色质组织的变化偶联，并允许对基因表达进行表观遗传控制。溴结构域通常存在于调节染色质结构和基因表达的蛋白质中，例如组蛋白乙酰转移酶和某些核小体重塑复合物的 ATP 酶成分。乙酰基的识别方式Bromo 结构域的赖氨酸类似于组蛋白乙酰转移酶的乙酰辅酶 A，尽管溴结构域是唯一已知与含有乙酰化赖氨酸的肽相互作用的结构域。溴结构域蛋白对乙酰赖氨酸的识别不限于组蛋白。例如，CREB ​​结合蛋白转录共激活因子 (CBP) 的溴结构域允许识别具有乙酰化 Lys382 的 p53。 bromodomain 和 acetyl-p53 之间的相互作用跟随 DNA 损伤并促进 p53 诱导的 CDK 抑制剂 p21 转录激活和细胞周期停滞。

Structure Reference Owen, D.J.等。 (2000) EMBO J. 19(22), 6141–6149。结构域蛋白示例结合位点 PCAF Tat BSYGRKAcKRRQRC CBP (CREB ​​Binding Protein) Ternary complex factor Elk-1 Not已知，SSPQPKKAcKPLDGE P53 SHLKSKKAcGQSTSRHKK，SSPQPKKAcKPLDGE Gcn5p 组蛋白 H4AKAcRHR Celtix-1 IRF-2 IRF-2 的超乙酰化形式显示更多HomeLearn & SupportProtein Domains & InteractionsEF-Hand Protein DomainEF-Hand Protein Domain

黑腹果蝇钙调蛋白中的 EF-Hand 结构域；红色的钙原子。

结构域结合和功能

EF-hand 基序包含大约 40 个残基，参与细胞内钙的结合。 EF-hand 结构域通常以单对或多对形式存在，从而导致包含 EF-hand 基序的蛋白质发生各种结构/功能变异。包含 EF 手的蛋白质可分为两个功能类别——调节和结构。钙与含有调节性 EF 手结构域的蛋白质的结合会引起构象变化，这种变化会传递给它们的靶蛋白，通常会催化酶促反应。相比之下，钙与结构 EF 手结构域的结合——包含蛋白质不会诱导显着的构象变化。结构性 EF-hand 结构域似乎在缓冲细胞内钙水平方面发挥作用。

结构参考 Taylor, D.A.等。 (1991) J. Biol。化学。 266（32），21375-80。结构域蛋白示例Proteins Binding Partners Functions Calmodulin Ca2+ 调节蛋白 S-100 Ca2+ 调节蛋白 Recoverin Ca2+ 调节蛋白 Calbindin Ca2+ 结构蛋白 Parvalbumin Ca2+ 结构蛋白 显示更多Western blot analysis of extracts from U118MG and CAD cells using PKA RI-α/β Antibody. Confocal immunofluorescent analysis of U-118 MG cells using PKA RI-α/β Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Western blot analysis of extracts from U118MG and CAD cells using PKA RI-α/β Antibody. Confocal immunofluorescent analysis of U-118 MG cells using PKA RI-α/β Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). 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.Western Blotting ProtocolFor 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 ReagentsFrom sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions KitNOTE: 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 dH2O, mix. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, 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 dH2O. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, 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 dH2O, mix. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, 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 BlottingA 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 l/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 l/lane) to determine molecular weights are recommended. Electrotransfer to nitrocellulose membrane (#12369).C. Membrane Blocking and Antibody IncubationsNOTE: Volumes are for 10 cm x 10 cm (100 cm2) 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 ProteinsDirections 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 2020A. Solutions and ReagentsAchieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions KitNOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies: Anti-Rabbit IgG (H+L), F(ab\')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 Anti-Rabbit IgG (H+L), F(ab\')2 Fragment (Alexa Fluor® 555 Conjugate) #4413 Anti-Rabbit IgG (H+L), F(ab\')2 Fragment (Alexa Fluor® 594 Conjugate) #8889 Anti-Rabbit IgG (H+L), F(ab\')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).B. Specimen Preparation - Cultured Cell Lines (IF-IC)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. Allow cells to fix for 15 min at room temperature. Aspirate fixative, rinse three times in 1X PBS for 5 min each. Proceed with Immunostaining (Section C).C. ImmunostainingNOTE: 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. Block specimen in Blocking Buffer for 60 min. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer. Aspirate blocking solution, apply diluted primary antibody. Incubate overnight at 4°C. Rinse three times in 1X PBS for 5 min each. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark. Rinse three times in 1X PBS for 5 min each. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961). For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light. Specificity / Sensitivity PKA RI-α/β Antibody detects endogenous levels of total PKA RI-α and PKA RI-β protein.Species Reactivity: Human, Mouse, Rat Species predicted to react based on 100% sequence homology: Pig Source / Purification Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asn310 of human PKA RI-α. Antibodies are purified by protein A and peptide affinity chromatography. Background The second messenger cyclic AMP (cAMP) activates cAMP-dependent protein kinase (PKA or cAPK) in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation (1). Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. Three C subunit isoforms (C-α, C-β, and C-γ) and two families of regulatory subunits (RI and RII) with distinct cAMP binding properties have been identified. The two R families exist in two isoforms, α and β (RI-α, RI-β, RII-α, and RII-β). Upon binding of cAMP to the R subunits, the autoinhibitory contact is eased and active monomeric C subunits are released. PKA shares substrate specificity with Akt (PKB) and PKC, which are characterized by an arginine at position -3 relative to the phosphorylated serine or threonine residue (2). Substrates that present this consensus sequence and have been shown to be phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3 (GSK-3α Ser21 and GSK-3β Ser9) (3-5). In addition, combined knock-down of PKA C-α and -β blocks cAMP-mediated phosphorylation of Raf (Ser43 and Ser259) (6). Autophosphorylation and phosphorylation by PDK-1 are two known mechanisms responsible for phosphorylation of the C subunit at Thr197 (7). Montminy, M. (1997) Annu. 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