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The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.
Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.
Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition1,2,3,4. The maintenance of low intraneuronal Cl- concentration ([Cl-]i) at 4-6 mM by KCC2 facilitates γ-aminobutyric acid (GABA)/glycine hyperpolarization and synaptic inhibition in the brain and spinal cord<....
NOTE: The protocol describes the western blotting method to detect specific proteins of interest.
1. Cell culture and transfection
Here, the representative result presented in Figure 1 investigated the impact of staurosporine and NEM on WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1 in HEK293 cell lines stably expressing KCC2b (HEKrnKCC2b)18 using the western blotting technique. Comprehensive details on the representative results are discussed in Zhang et al.15. Similar to NEM, staurosporine is a broad kinase inhibitor that can enhance KCC2 tr.......
Many methods have been used to measure the activities of SLC12 of CCCs that are expressed in the neurons, including KCC2. Many of these techniques have proven to enhance scientific knowledge on the analysis of the functional relevance of these transporters and their structure-function patterns in different disease-related mutations. Critically, there are advantages and caveats to the various methods21. However, the protocol explained above, outlined how to assess KCC2 phosphorylation at kinase reg.......
This work was supported by The Royal Society UK (Grant no. IEC\NSFC\201094), and a Commonwealth Ph.D. Scholarship.
....Name | Company | Catalog Number | Comments |
40% acrylamide | Sigma-Aldrich | A2917 | Used to make seperating and stacking gel for SDS-PAGEÂ |
Ammonium Per Sulfate | Sigma-Aldrich | 248614 | Used to make seperating and stacking gel for SDS-PAGEÂ |
anti pSPAK | Dundee University | S670B | Used as primary antibody for western blotting |
anti-KCC2 | Dundee University | S700C | Used as primary antibody for western blotting |
anti-KCC2 pSer940 | Thermo Fisher Scientific | PA5-95678 | Used as primary antibody for western blotting |
anti-KCC2 pThr1007 | Dundee University | S961C | Used as primary antibody for western blotting |
anti-KCC2 pThr906 | Dundee University | S959C | Used as primary antibody for western blotting |
anti-mouse | Cell Signalling technology | 66002 | Used as secondary antibody for western blotting |
anti-NKCC1 | Dundee University | S841B | Used as primary antibody for western blotting |
anti-NKCC1 pThr203/207/212 | Dundee University | S763B | Used as primary antibody for western blotting |
anti-rabbit | Cell Signalling technology | C29F4 | Used as secondary antibody for western blotting |
anti-sheep | abcam | ab6900 | Used as secondary antibody for western blotting |
anti-SPAK | Dundee University | S669D | Used as primary antibody for western blotting |
anti-β-Tubulin III | Sigma-Aldrich | T8578 | Used as primary antibody for western blotting |
Benzamine | Merck UK | 135828 | Used as component of lysis buffer |
Beta-mercaptoethanol | Sigma-Aldrich | M3148 | Used as component of loading buffer and lysis buffer |
Bradford Coomasie | Thermo Scientific | 1856209 | Used for lysate protein quantification |
Casting apparatus | Atto | WSE-1165W | Used to run SDS-page electrophoresis |
Centrifuge | Eppendorf | 5804 | Used in lysate preparation |
Centrifuge | VWR | MicroStar 17R | Used for spinning samples |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650-100ML | Used for cell culture experiment |
Dried Skimmed Milk | Marvel | N/A | Used to make blocking buffer |
Dulbecco's Modified Eagle's Medium - high glucose | Sigma-Aldrich | D6429 | Used for cell culture |
ECL reagent | Perkin Elmer | ORTT755/2655 | Used to develop image for western blotting |
EDTA | Fisher Scientific | D/0700/53 | Used as component of lysis buffer |
EGTA | Sigma-Aldrich | e4378 | Used as component of lysis buffer |
Electrophoresis Power Supply | BioRad | PowerPAC HC | To supply power to run SDS-page electrophoresis |
Ethanol | ThermoFisher | E/0650DF/17 | Used for preparing sterilized equipments and environment |
Fetal Bovine Serum -Â heat inactivated | Merck Life Sciences UK | F9665 | Used for cell culture |
Fumehood | Walker | A7277 | Used for cell culture |
Gel Blotting - Whatman | GE Healthcare | 10426981 | Used in western blotting to make transfer sandwich |
Glycine | Sigma-Aldrich | 15527 | Used to make buffers |
GraphPad Prism Software | GraphPad Software, Inc., USA | Version 6.0 | Used for plotting graphs and analysing data for western blotting |
HCl | Acros Organics | 10647282 | Used to make seperating and stacking gel for SDS-PAGEÂ |
Heating block | Grant | QBT1 | Used to heat WB loading samples |
HEK293 cells | Merck UK | 12022001-1VL | Cell line for culture experiment |
ImageJ Software | Wayne Rasband and Contributors; NIH, USAÂ | ImageJ 1.53e | Used to measure band intensities from western blotting images |
Imaging system | BioRad | ChemiDoc MP | Used to take western blotting images |
Incubator | LEEC | LEEC precision 190D | Used for cell culture |
Isopropanol | Honeywell | 24137 | Used in casting gel for electrophoresis |
L-glutamine solution | Sigma-Aldrich | G7513 | Used for cell culture |
Lithium dodecyl sulfate (LDS) | Novex | NP0008 | Used as loading buffer for western blotting |
MEM Non-essential amino acid | Merck Life Sciences UK | M7145 | Used for cell culture |
Microcentrifuge | Eppendorf | 5418 | Used for preparing lysates for WB |
Microplate reader | BioRad | iMark | Used for lysate protein concentration readout |
Microsoft Powerpoint | Microsoft, USA | PowerPoint2016 | Used to edit western blotting images |
Molecular Weight Marker | BioRad | 1610373 | Used for western blotting |
N-ethylmaleimide | Thermo Fisher Scientific | 23030 | Used for cell culture experiment |
Nitrocellulose membrane | Fisher Scientific | 45004091 | Used for western blotting |
Penicillin-Streptomycin | Gibco | 15140122 | Used for cell culture |
pH Meter | Mettler Toledo | Seven compact s210 | Used to monitor pH of buffer solutions |
Phenylmethylsulfonylfluoride (PMSF) | Sigma-Aldrich | P7626 | Used as component of lysis buffer |
Phosphate Buffer Saline | Sigma-Aldrich | D8537 | Used for cell culture |
PKCδ pThr505 | Cell Signalling technology | 9374 | Used as primary antibody for western blotting |
Sepharose Protein G | Generon | PG50-00-0002 | Used for immunoprecipitation |
Sodium chloride | Sigma-Aldrich | S7653 | Used as component of wash buffer |
Sodium Chloride | Sigma-Aldrich | S7653 | Used to prepare TBS-T buffer |
Sodium Dodecyl Sulfate | Sigma-Aldrich | L5750 | Used to make seperating and stacking gel for SDS-PAGEÂ |
sodium orthovanadate | Sigma-Aldrich | S6508 | Used as component of lysis buffer |
Sodium Pyruvate | Sigma-Aldrich | S8636 | Used for cell culture |
sodium-β-glycerophosphate | Merck UK | G9422 | Used as component of lysis buffer |
Staurosporine (from Streptomyces sp.) | Scientific Laboratory Supplies, UK | S4400-1MG | Used for cell culture experiment |
Sucrose | Scientifc Laboratory Supplies | S0389 | Used as component of lysis buffer |
TEMED | Sigma-Aldrich | T7024 | Used to make seperating and stacking gel for SDS-PAGEÂ |
Transfer Chamber | BioRad | 1658005EDU | Used in western blotting to transfer protein on membrane |
Tris | Sigma-Aldrich | T6066 | Used to make seperating and stacking gel for SDS-PAGEÂ |
Triton-X100 | Sigma-Aldrich | T8787 | Used as component of lysis buffer |
Trypsin-EDTA Solution | Merck Life Sciences UK | T4049 | Used for cell culture |
Tween-20 | Sigma-Aldrich | P3179 | Used as make TBS-T buffer |
Vacuum pump | Charles Austen | Dymax 5 | Used for cell culture |
Vortex | Scientific Industries | K-550-GE | Used in sample preparation |
Vortex mixer | Scientific Industries Ltd | Vortex-Genie K-550-GE | Used of mixing resolved sample |
Water bath | Grant Instruments Ltd. (JB Academy) | JBA5 | Used to incubate solutions |
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