Pleckstrin Homology (PH) Domains are small, conserved protein domains that bind to phosphoinosit1des and play a key role in various cellular processes, such as signal transduction, cytoskeleton organization, and membrane trafficking.
These domains are found in a diverse array of proteins and are important for their localization and regulation.
PH Domains typically consist of approximately 120 amino acids and adopt a distinctive beta-sandwich fold with a C-terminal alpha-helix.
They can bind to various phosphoinositide species, including PIP2 and PIP3, which helps recruit the host proteins to specific membrane compartments.
PH Domains are crucial for a variety of cellular functions, including cell growth, survival, and motility, and their dysregulation has been implicated in various disease states, making them an important target for biomedical research and drug development.
Most cited protocols related to «Pleckstrin Homology Domains»
The PH domains of PLCδ1 (1–170), Bruton's tyrosine kinase (1–177), Akt protein kinase (1–167), and dynamin (508–652) were amplified with the Advantage Klentaq polymerase mix (CLONTECH Labs, Inc., Palo Alto, CA) from human cDNAs (marathon cDNA from brain and K562 leukemia cells; CLONTECH Labs, Inc.) with the following primer pairs: PLCδ: 5′-GGCATGGACTCGGGCCGGGACTTCCTG-3′, 5′-AAGATCTTCCGGGCATAGCTGTCG-3′; Btk: 5′-CCAAGTCCTGGCATCTCAATGCATCTG-3′, 5′-TGGAGACTGGTGCTGCTGCTGGCTC-3′; Akt: 5′-GTCAGCTGGTGCATCAGAGGCTGTG-3′, 5′-CACCAGGATCACCTTGCCGAAAGTGCC-3′; Dyn: 5′-ATGCTCAGCAGAGGAGCAACCAGATG-3′, 5′-GAGTCCACAAGATTCCGGATGGTCTC-3′. The amplified products were subcloned into the PGEM-Easy T/A cloning vector (Promega Corp., Madison, WI) and sequenced with dideoxy sequencing (thermosequenase; Amersham Corp.). A second amplification reaction was performed from these plasmids with nested primers that contained restriction sites for appropriate cloning into the pEGFP-N1 (PLCδ, Btk, and Akt) or pEGFP-C1 (dynamin) plasmids (CLONTECH Labs, Inc.) to preserve the reading frame. Plasmids were transfected into COS-7 cells or NIH-3T3 cells and cell lysates were resolved by SDS-PAGE followed by Western blot analysis for the presence of the GFP fusion proteins using a polyclonal antibody against GFP (CLONTECH Labs, Inc.). Mutations were created in the PHPLCδ–GFP fusion plasmid by the QuickChange™ mutagenesis kit (Stratagene, La Jolla, CA). For practical purposes, a SalI site was introduced into the PH domain sequence which changed S34 to a T but this substitution did not change any characteristic compared with the wild-type protein. All mutations were confirmed by dideoxy sequencing and the expression of the fusion protein by Western blot analysis.
Várnai P, & Balla T. (1998). Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools. The Journal of Cell Biology, 143(2), 501-510.
Experimental data were collected on three different proteins: (1) a 1 mM sample of the 8 kDa CUE domain containing residues 453–504 from human gp78 (Liu et al. 2012 (link)); (2) a 330 μM sample of the 15 kDa PH domain of ASAP1, which contains residues 339–451 (Luo et al. 2008 (link)); and (3) a 400 μM sample of the 32 kDa two domain construct (ZA) of ASAP1 containing residues 441–724 (Luo et al. 2008 (link)). Isotope labeling was performed by expressing and purifying the proteins from E. coli using standard techniques to produce either uniform 13C, 15N-labeled protein, uniform 2H, 13C, 15N-labeled protein (DCN), or uniform 2H, 13C, 15N, 13C1H3-methyl (Ileδ1, Leu, Val) labeled protein (DCN-ILV) or 2H, 15N, 13C1H3-methyl (Ileδ1, Leu, Val) labeled protein (DCmethylN-ILV). NESTA-NMR has been used to process a wide range of 3D and 4D NMR experiments that were collected on these three samples, and the salient information of all of these experiments is listed in Supplemental Table 1. Data discussed explicitly in the manuscript consist of the following four data sets:
A 4D methyl-methyl HMQC-NOESY-HMQC experiment (4D CC-NOESY) utilizing mixed constant-time evolution (Ying et al. 2007 (link)) was recorded on 1 mM DCNILV gp78 CUE using a Bruker Avance 900 MHz instrument running TopSpin 2 with cryoprobe at 298K. The standard pulse sequence was modified to store all of the hypercomplex pairs adjacent to each other with quadrature modulations preceding time modulation and the delays in the indirect dimensions calculated according to a NUS sampling schedule. In order to compare reconstructions with those of different programs, the sampling schedule was produced by an in-house Python script according to the algorithm described by Mobli et al. (Mobli et al. 2010 (link)) which was additionally modified to ensure every index for a given dimension contained at least one sampling point. Sampling consisted of 7200 NUS points taken on a 48 13 C × 32 1H × 48 13C grid with a sampling density of 9.8%. In this report, the number of points of an indirect dimension is described in complex points—i.e. real and imaginary data are counted as one point. The maximum evolution times in the indirect dimensions were 11.5 ms for both 13C dimensions and 34.1 ms for the indirect 1H dimension. Spectral widths were 4098 Hz for both 13C dimensions and 909 Hz for the indirect 1H dimension. Each FID was recorded with 4 scans, and the NOE mixing period was 150 ms.
A variable (non-constant) time 4D methyl-methyl HMQC-NOESY-HMQC (Diercks et al. 1999 (link)) experiment was acquired on a 400 μM sample of DCmethylN-ILV ZA on a Bruker Avance III 600 MHz instrument with cryoprobe at 298K using TopSpin 3.2. The sampling schedule was designed with ScheduleTool, which is distributed with RNMRTK (Hoch and Stern 1996 ), and consisted of 12,000 NUS points taken on a 48 13 C × 64 1 H × 48 13C grid with a sampling density of 8.1%. The maximum evolution times in the indirect dimensions were 12.2 ms for both 13C dimensions and 19.0 ms for the indirect 1H dimension. Spectral widths were 3922 Hz for both 13C dimensions and 3360 Hz for the indirect 1H dimension. Each FID was recorded with 4 scans and the NOE mixing period was 200 ms.
Two 3D 15N-edited NOESY-HSQC experiments were acquired on a 330 μM 15N-labeled PH domain on a Bruker Avance III 600 MHz instrument with cryoprobe at 298 K using the Topspin 3.2 library pulse sequence nosesyhsqcf3gp193d (Sklenar et al. 1993 ). One data set was collected with uniform sampling (36 13 C × 180 1H) and serves as the reference. The other was collected with 1620 NUS points (25% sampling density) on a 36 × 180 grid. The sampling schedule was designed with ScheduleTool. For both experiments, the maximum evolution times in the indirect dimensions were 18.5 ms for 15N and 25 ms for 1H. Spectral widths were 1945 Hz for 15N and 7194 Hz for 1H. The NOE mixing period was 60 ms. Each FID contained 8 and 32 scans for the uniformly sampled and non-uniformly sampled data, respectively.
Data reconstruction was performed using in-house C programs for both the NESTA algorithm and alternative algorithms used for comparison. This was done to enable direct comparison of convergence rates since package-specific implementations may affect computing efficiency. Thus, all the algorithms utilized the same libraries and were compiled on the same computer. Mixed-radix FFT and IFFT routines from the GNU Scientific Library (GSL) (Galassi et al. 2009 ) capable of processing complex vectors of any length (not restricted to powers of 2) were used to construct multidimensional subroutines to transform hypercomplex data. Direct comparison of algorithms rather than a specific software package is enabled because the algorithms utilize the same libraries and the analysis of computational efficiency is measured by the number of iterations required to reach convergence. The processing package NESTA-NMR was developed to apply NESTA minimization to 2D, 3D, and 4D NMR data. Data described in this manuscript were processed on a desktop computer running Centos 6 with a 2.13 GHz Intel Xeon processor containing 4 hyperthreaded cores (8 threads) or a Mac Pro with a 3.5 GHz Intel Xeon processor containing 6 hyperthreaded cores (12 threads). The software can also be run on a cluster to access even more threads; however, this is not generally necessary given the relatively short computational times of NESTA-NMR, even for 4D data (vide infra). After reconstructing the unsampled data points and merging them with experimentally sampled data, the indirect dimensions were processed with NMRPipe (Delaglio et al. 1995 (link)) using standard FFT methods for transformation and visualized using Sparky (Goddard and Kneller).
Sun S., Gill M., Li Y., Huang M, & Byrd R.A. (2015). Efficient and generalized processing of multidimensional NUS NMR data: the NESTA algorithm and comparison of regularization terms. Journal of biomolecular NMR, 62(1), 105-117.
Previously, we characterized the gene encoding mouse α-syntrophin using a genomic library derived from 129Sv DNA (Adams et al. 1995). A targeting vector was constructed by cloning a 7.4-kb NotI/XbaI restriction fragment from the region 5′ of exon 1 and a 1.5-kb XbaI fragment from intron 1 into the plasmid JNS2 (Dombrowicz et al. 1993; Fig. 1). The resulting recombinant gene is missing the entire first exon (amino acids 1–97, which encode half of the first PH domain and part of the PDZ domain), 0.8 kb of 5′ flanking sequence, and 1.7 kb of the first intron. The targeting vector was linearized with NotI and transferred to E14 ES cells by electroporation. After selection with G418 and gancyclovir (a gift from Roche Biosciences), homologous recombinant ES cells were identified by Southern blotting (Fig. 1) and used for C57Bl6 blastocyst injection. Injections were performed by the UNC-CH Embryonic Stem Cell Facility. Chimeras were bred with C57Bl6 females and germline transmission was confirmed by Southern blot analysis and PCR (primers: neo-CAAATTAAGGGCCAGCTCATTCCTCC; α-syntrophin first intron-ACAGGAGCCCAGTCTTCAATCCAGG). The mice used in this study were adults >10-wk old and either first generation with a mixed 129Sv/C57Bl6 background or had been bred back against C57Bl6 for 3 generations. Mice with either background show similar phenotypes.
Adams M.E., Kramarcy N., Krall S.P., Rossi S.G., Rotundo R.L., Sealock R, & Froehner S.C. (2000). Absence of α-Syntrophin Leads to Structurally Aberrant Neuromuscular Synapses Deficient in Utrophin. The Journal of Cell Biology, 150(6), 1385-1398.
AKT1-Inhibitor VIII co-crystals were harvested into a solution of 25 mM Na-acetate, 25 mM Na-citrate, 21% PEG MME 2000, pH 5.0 and were cryoprotected with 70% harvest solution + 30% ethylene glycol. Cryoprotected crystals were flash cooled in a stream of dry nitrogen vapor held at 100 K. X-ray diffraction data were collected on a Rigaku FR-E Superbright rotating anode X-ray generator, fitted with a Cu anode and an RAXIS IV++ image plate detector (Rigaku, TX, USA). The diffraction data were processed using Mosflm [26] and scaled using the program Scala [27] (link). The crystals belonged to space group P21 with unit cell dimensions of a = 49.31 Å, b = 69.94 Å, c = 61.85 Å, β = 100.6°. The crystal structure was solved by molecular replacement, with all calculations performed using the program Molrep [27] (link). The molecular replacement calculations were performed in two steps: In the first step, a search model consisting of residues 147–440 of the inactive AKT2 kinase domain (PDB code: 1MRV) was used in a standard rotation function/translation function calculation, resulting in a single solution with an R-factor of 0.505 (similar searches with an active conformation of AKT1 kinase domain failed to find a reliable solution). The quality of this molecular replacement solution was improved slightly by brief crystallographic refinement to 2.8 Å resolution in Refmac5. In the second step, a search model consisting of residues 2–106 of the unliganded AKT1 PH domain (PDB code: 1UNP) was used in a rotation function/phased translation function search, using phase information calculated from the coordinates of the AKT2 kinase domain solution. A single PH domain plus kinase domain solution was found with an R-factor of 0.417. This combined solution was subjected to multiple cycles of refinement in Refmac5 to 2.7 Å resolution [28] (link), followed by model rebuilding in the program O [29] (link). The final round of model rebuilding was guided by the use of simulated annealing composite omit maps followed by crystallographic refinement in CNX 2005 [30] (link) to generate the final molecular model. The final structure contains all residues of AKT1 from 2–429 except for 45–48, 114–144 (the linker region), 189–198 (the αB and αC helices) and 299–312 (the C-terminal end of the activation loop). The model also has 21 ordered water molecules and a single copy of Inhibitor VIII. The R-factor of the final model is 0.245 with an Rfree value of 0.307. 312 residues (98.7%) lie in the “most-favored” or “additional” regions of the Ramachandran plot, with 2 residues (0.6%) in the “generously allowed” region and 2 (0.6%) in the “disallowed” regions. All figures were generated using Pymol [31] .
Wu W.I., Voegtli W.C., Sturgis H.L., Dizon F.P., Vigers G.P, & Brandhuber B.J. (2010). Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition. PLoS ONE, 5(9), e12913.
A plasmid encoding Rluc8 was provided by Dr. Sanjiv Sam Gambhir, and a plasmid containing the PH domain of PLC δ1 was provided by Dr. Tobias Meyer (both at Stanford University, Palo Alto, CA). A plasmid encoding venus-K-Ras was provided by Dr. Stephen R. Ikeda (NIAAA, Rockville, MD). Plasmids encoding giantin, MoA and an ER-targeted FKBP (CFP-FKBP-Cb5) were provided by Dr. Takanari Inoue (Johns Hopkins University, Baltimore, MD). Plasmids encoding RIT and H-Ras were provided by Dr. Mark R. Philips (New York University, New York, NY). Plasmids encoding CD3δ were provided by Dr. Jennifer Lippincott-Schwartz (NICHD, Bethesda, MD). All other constructs were made using an adaptation of the QuikChange (Stratagene) mutagenesis protocol and were verified by automated sequencing.
Lan T.H., Liu Q., Li C., Wu G, & Lambert N.A. (2012). Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET. Traffic (Copenhagen, Denmark), 13(11), 1450-1456.
Fig. S1 shows the interaction between Tex2 and E-Syt1 by coIP assays and microscopy, and also shows the information about the GFP-Tex2 knock-in and Tex2 knockout cell lines by Crispr-cas9 technology. Fig. S2 provides additional data to support that Tex2 is a tubular ER protein and its targeting to tubular ER is independent of ER tubule-shaping proteins. Fig. S3 contains additional data to show the role of Tex2 in the formation of ER tubules and also provide data to show that Tex2 may not be directly involved in TG-induced ER stress. Fig. S4 provides additional data to show that either the PH domain or the SMP domain of Tex2 is not required for Tex2 recruitment to ER–LE/lys MCSs by TMEM55B. Fig. S5 contains additional data to show the role of Tex2 in lysosomal function and the distribution of PS, PI4P, and PI(4,5)P2. Video 1 shows an example of a lysosome undergoing retrograde transport in a COS7 cell transfected with an ER marker and a LE/lys marker. Video 2 shows an example of a Halo-TMEM55B-positive lysosome tightly associating with GFP-Tex2-labeled ER membranes during intracellular transport in a COS7 cell.
Du Y., Chang W., Gao L., Deng L, & Ji W.K. (2023). Tex2 is required for lysosomal functions at TMEM55-dependent ER membrane contact sites. The Journal of Cell Biology, 222(4), e202205133.
We searched the BLAST non-redundant protein sequence database for amphibian PLCZ1 orthologs using the protein sequence of mouse Plcz1 as a query. We retrieved annotated orthologs from six amphibian species and included them, along with sequences encoding other PLC isoforms (beta, gamma, delta, epsilon, and eta) in an alignment of representative PLC sequences from amphibians, birds, and mammals using Kalign (Lassmann, 2020 ). We used the neighbor-joining phylogenetic output from this alignment to generate an unrooted tree with Dendroscope (Huson and Scornavacca, 2012 (link)). We expanded our search for PLCZ1 orthologs by performing a reverse translated tblastn search using the sequence of the Nanorana parkerii PLCZ1 as a query against the amphibian transcriptome shotgun assembly (TSA) database. We retrieved more than 100 sequences of possible PLCZ1 orthologs, which we translated using getorf (EMBOSS), selecting the longest open reading frames from each transcript. We aligned these sequences with the representative PLC sequences that we had previously obtained from amphibians, birds, and mammals to distinguish genuine PLCZ1 orthologs from hits that encoded other PLC subtypes. Through this analysis, we identified genes encoding PLCZ1 in 5 additional amphibian species. Finally, we used InterProScan (Nomikos et al., 2005 (link)) to validate the potential PLCZ1 orthologs that we had identified by looking for the absence of a pleckstrin homology (PH) domain, which is a defining feature of PLCζ enzymes.
Bainbridge R.E., Rosenbaum J.C., Sau P, & Carlson A.E. (2023). Xenopus laevis lack the critical sperm factor PLCζ. bioRxiv.
The PH-Like 1 domain of BoHV-1 gB was fused, via 8X Gly-Ser linker to the PH-Like 2 domain of BoHV-5 gB. The 3D structure of this fused protein was predicted with the same server used. The candidate construct (gBDomains) was evaluated for conformational and linear epitopes for B lymphocytes using Ellipro [45 (link)] and BCPreds [46 (link)], respectively. Epitopes for T lymphocytes were also predicted using the EpiJen [47 (link)] for the candidate construct.
Quintero Barbosa J.S., Rojas H.Y., Gonzalez J., Espejo-Mojica A.J., Díaz C.J, & Gutierrez M.F. (2023). Characterization and expression of domains of Alphaherpesvirus bovine 1/5 envelope glycoproteins B in Komagataella phaffi. BMC Veterinary Research, 19, 28.
Generation of rAd-expressing shRNA-targeting human or mouse Myo1b driven by the U6 promoter (rAd/U6-hMyo1b shRNA or rAd/U6-mMyo1b shRNA) was carried out with the Gateway Technology. The targeting sequences are indicated in underline below [18 (link)]: hMyo1b-shRNA: 5′-CACCGGGCTTTATGGATCATGAAGCCGAAGCTTCATGATCCATAAAGCCC-3′. mMyo1b-shRNA: 5′-CACCGGAGCTCCTCTACAAGCTTAACGAATTAAGCTTGTAGAGGAGCTCC-3′. rAd/U6-LacZ shRNA served as control was generated as previously described [18 (link)]. The purification and mouse tail vein injection of rAd/U6-mMyo1b shRNA adenovirus particles were performed as described by Tan et al. [26 (link)]. Generation of rAd expressing myc-MYO1B-WT and its mutants -R165A (deficient in its motor activity) and -K966A (deficient in its C-terminal PH domain) driven by CMV promoter (rAd/CMV-myc-MYO1B-WT, -R165A, and -K966A) was also carried out with the Gateway Technology. The expression plasmids encoding myc-MYO1B-WT, -R165A, and-K966A were kindly provided by Lynne M. Coluccio [17 (link)].
Yu Y., Ren Y., Li Z., Li Y., Li Y., Zhang Y., Gui R., Cui Y., Qian L, & Xiong Y. (2023). Myo1b Promotes Premature Endothelial Senescence and Dysfunction via Suppressing Autophagy: Implications for Vascular Aging. Oxidative Medicine and Cellular Longevity, 2023, 4654083.
Atomic coordinates for starting structures were acquired from the Protein Data Bank (85 (link)): 1W1G was used for the PDK1 PH domain (39 (link)) and 4RRV (31 (link)) was used for the PDK1 catalytic domain. Molecular dynamics (MD) simulations of the PH domain bound to PIP3 were carried out with pmemd.cuda from AMBER18 (86 (link)–88 ) using AMBER ff14SB force field (89 (link)) and generalized amber force field (GAFF) (90 (link)). We used tLeap binary (part of AMBER18) for solvating structures in a cubed TIP3P water box with a 10 Å distance from structure surface to the box edges and closeness parameter of 0.75 Å. The system was neutralized and solvated. Simulations were carried out after minimizing the system, gradually heating the system from 0 K to 300 K over 50 ps and equilibrating the system for 1 ns at NPT. 500 ns of production was carried out using NPT at 300 K with the Langevin thermostat, a non-bonded interaction cut off of 8 Å, time step of 2 fs, and the SHAKE algorithm to constrain all bonds involving hydrogens. RMSD calculations were done with VMD (91 (link)) to analyze fluctuations in the structural ensemble to determine stability; stabilized regions close to the linker were then docked to the catalytic domain using ClusPro 2.0 (92 (link)–94 (link)) to form initial configurations of full-length PDK1. Molecular dynamics simulations of the PH domain bound to PIP3 showed allosteric changes in stability in regions close to the linker region, namely in the helix at 434-443. This helix was then used for docking to the catalytic domain using ClusPro2.0 (92 (link), 93 (link)) to give an orientation for the PH-catalytic domain interface. The helix at position 372-381 was modeled from the helix of the pseudosubstrate inhibitor peptide PKI (5-24) TTYADFIASGRTGRR bound to PKA from 1CDK (57 (link)), with Tyr7 and Phe10 matching with Tyr373 and Tyr376, respectively. The homology model of the helix was constructed by making point mutations using PyMOL. The helix at position 395-400 was docked to the catalytic domain with ClusPro2.0 and the remainder of the linker was built using the Builder in Pymol. The model was minimized and equilibrated using MD simulations similar to the above but using the implicit solvent forcefield GBNeck2 (95 (link)) igb=8 with PBRadii set to mbondi3. Finally, we simulated this model for five hundred nanoseconds and the PH domain stayed in the same pocket with both flanking helices 372-381 and 395-400 remaining bound to the catalytic domain. This points to having found a robust binding mode because weak docked poses will often leave their binding site during simulation.
Sacerdoti M., Gross L.Z., Riley A.M., Zehnder K., Ghode A., Klinke S., Anand G.S., Paris K., Winkel A., Herbrand A., Godage H.Y., Cozier G.E., Süß E., Schulze J.O., Pastor-Flores D., Bollini M., Cappellari M.V., Svergun D., Gräwert M.A., Aramendia P.F., Leroux A.E., Potter B.V., Camacho C.J, & Biondi R.M. (2023). Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein. Science signaling, 16(789), eadd3184.
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The Amaxa Nucleofector II is a laboratory instrument designed for the efficient and gentle transfection of various cell types, including difficult-to-transfect cells. It utilizes an electroporation-based technology to introduce nucleic acids, such as plasmids, siRNA, or mRNA, into the target cells.
The PEGFP-C1 vector is a plasmid-based expression vector that enables the production of proteins fused with the enhanced green fluorescent protein (EGFP) tag. The vector contains the cytomegalovirus (CMV) promoter for high-level expression in mammalian cells, as well as the gene encoding the EGFP protein for visualization and detection purposes.
Atto 532 is a fluorescent dye with an absorption maximum at 532 nm and an emission maximum at 553 nm. It is a member of the Atto dye family developed by the Atto-Tec GmbH company, which is now part of the Merck Group. The Atto 532 dye is designed for use in various fluorescence-based applications, such as microscopy, flow cytometry, and biochemical assays.
AKT-PH-GFP is a fluorescent protein construct that contains the pleckstrin homology (PH) domain of the AKT protein fused to green fluorescent protein (GFP). This construct can be used to visualize the localization of AKT in live cells.
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The PGEX-4T-1 is a laboratory equipment product designed for protein expression. It functions as a vector for the expression and purification of recombinant proteins in Escherichia coli (E. coli) host cells.
PIP strips are a laboratory tool used for the detection and identification of phosphoinositides (PIs) in biological samples. These strips contain a series of immobilized lipids that can be used to analyze the presence and relative abundance of different PI species through a simple immunoblotting procedure.
The PGEX-6P-1 vector is a plasmid system used for the expression and purification of recombinant proteins in Escherichia coli. It features a tac promoter for high-level protein expression, a GST (Glutathione S-Transferase) tag for affinity purification, and a PreScission Protease cleavage site for tag removal.
Alexa Fluor 647 is a fluorescent dye that can be used for labeling and detection of biological molecules. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the far-red to near-infrared region of the spectrum.
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The ChemiDoc Imaging System is a versatile lab equipment designed for the visualization and analysis of various biomolecules, such as proteins, nucleic acids, and chemiluminescent samples. It utilizes advanced imaging technologies to capture high-quality digital images for downstream applications.
Glutathione agarose is a chromatography resin used for the purification of glutathione S-transferase (GST) fusion proteins. It consists of reduced glutathione that is covalently coupled to agarose beads. The glutathione serves as a ligand that specifically binds to GST, allowing the target protein to be isolated from complex mixtures.
Pleckstrin Homology (PH) Domains are small, conserved protein domains that bind to phosphoinositides and play a key role in various cellular processes, such as signal transduction, cytoskeleton organization, and membrane trafficking. These domains typically consist of approximately 120 amino acids and adopt a distinctive beta-sandwich fold with a C-terminal alpha-helix. PH Domains can bind to different phosphoinositide species, including PIP2 and PIP3, which helps recruit the host proteins to specific membrane compartments. This localization is crucial for a variety of cellular functions, including cell growth, survival, and motility.
Pleckstrin Homology Domains can be found in a diverse array of proteins and exhibit some variations in their structure and binding preferences. While most PH Domains share a common fold, they can differ in their specificity for different phosphoinositide species. Some PH Domains preferentially bind to PIP2, while others may have a higher affinity for PIP3. There are also PH Domains that can bind to other lipids or even proteins, expanding their functional roles within the cell.
PubCompare.ai can be a valuable tool for optimizing Pleckstrin Homology Domain research. The platform allows you to more efficiently screen protocol literature, leveraging AI to pinpoint critical insights. PubCompare.ai's AI-driven analysis can help researchers identify the most effective protocols related to Pleckstrin Homology Domains for their specific research goals. By highlighting key differences in protocol effectiveness, the platform enables you to choose the best option for reproducibility and accuracy, streamlining your Pleckstrin Homology Domain research.
One of the main challenges in working with Pleckstrin Homology Domains is their diverse functions and binding preferences. Since PH Domains can interact with a variety of phosphoinositides and other biomolecules, it can be difficult to predict their exact role in a given cellular context. Additionally, the subtle structural differences between PH Domains can lead to variations in their binding affinities and specificities, requiring careful optimization of experimental conditions. Researchers must also be mindful of potential cross-reactivity or non-specific interactions when using PH Domains as tools for lipid or protein detection and localization.
PubCompare.ai can help address the challenges in Pleckstrin Homology Domain research by providing a platform for more efficient protocol screening and AI-driven analysis. The tool can assist researchers in identifying the most effective and reproducible protocols for working with PH Domains, taking into account their diverse functions and binding properties. By leveraging AI to compare protocols from the literature, pre-prints, and patents, PubCompare.ai can highlight the critical differences that may impact experimental outcomes, allowing researchers to make more informed decisions about the best approaches for their specific research goals. This can help overcome the challenges posed by the structural and functional complexity of Pleckstrin Homology Domains and streamline the research process.
More about "Pleckstrin Homology Domains"
Pleckstrin Homology (PH) Domains are a family of small, highly conserved protein domains that play a crucial role in various cellular processes.
These modular structures, typically consisting of around 120 amino acids, adopt a distinctive beta-sandwich fold with a C-terminal alpha-helix.
PH Domains are known for their ability to bind to phosphoinositides, a class of lipid messengers, including PIP2 and PIP3, which helps recruit the host proteins to specific membrane compartments.
PH Domains are found in a diverse array of proteins, including those involved in signal transduction, cytoskeleton organization, and membrane trafficking.
Their binding to phosphoinositides is essential for the localization and regulation of these proteins, making them key players in cell growth, survival, and motility.
Dysregulation of PH Domains has been implicated in various disease states, highlighting their importance as targets for biomedical research and drug development.
Researchers often utilize tools and techniques to study PH Domains, such as the Amaxa Nucleofector II for transfection, the PEGFP-C1 vector for fluorescent protein tagging, and the Atto 532 dye for labeling.
The AKT-PH-GFP construct is a common tool for visualizing PH Domain localization, while the PGEX-4T-1 and PGEX-6P-1 vectors are used for protein expression and purification.
PIP strips, a membrane-based assay, can be employed to assess the binding specificity of PH Domains.
The ChemiDoc Imaging System is often used for imaging and quantifying protein signals, and Glutathione agarose is a common resin for affinity purification of GST-tagged PH Domain proteins.
By leveraging these tools and techniques, researchers can gain insights into the structure, function, and regulation of Pleckstrin Homology Domains, ultimately contributing to our understanding of their role in cellular processes and their potential as therapeutic targets.
