immunoblot

fixa-idea asked:

Or if that one was too obvious, Southern Blotting?

Western blotting. I think southern blotting is a thing, but I’ve never done it.

IT’S JUST REALLY COOL in terms of the science that’s involved. You separate your proteins by electricity and then stick antibodies onto antibodies that you have stuck to proteins and THEN you stick a glowy substrate onto THAT and then you can see only the protein you want! And then measure the intensity of your proteins to see what your cell culture treatments did! AND THEN YOU THROW YOUR APPARATUS ACROSS THE ROOM BECAUSE THE TREATMENTS HAD THE OPPOSITE EFFECT THAN YOUR HYPOTHESIS AND OMG I’LL NEVER GET A PAPER OUT OF THIS AND THEN YOU REPEAT UNTIL YOU DIE.

Homozygous splice mutation in CWF19L1 in a Turkish family with recessive ataxia syndrome.

PubMed:
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Homozygous splice mutation in CWF19L1 in a Turkish family with recessive ataxia syndrome.

Neurology. 2014 Dec 2;83(23):2175-82 

Authors: Burns R, Majczenko K, Xu J, Peng W, Yapici Z, Dowling JJ, Li JZ, Burmeister M

Abstract
OBJECTIVE: To elucidate the genetic cause of a rare recessive ataxia presented by 2 siblings from a consanguineous Turkish family with a nonprogressive, congenital ataxia with mental retardation of unknown etiology.
METHODS: Whole-exome sequencing was combined with homozygosity mapping, linkage, and expression analysis to identify candidate genes, confirmed by Sanger sequencing. Reverse transcription-PCR and immunoblotting were used to determine the functional consequences of the gene variant. A zebrafish model was developed using morpholino-mediated knockdown.
RESULTS: We identified a homozygous mutation at the invariant +1 position (c.964+1G>A) in intron 9 of the CWF19L1 (complexed with cdc5 protein 19-like 1) gene. This mutation is absent in >6,500 European and African American individuals and 200 Turkish control DNAs. The mutation causes exon skipping, reduction in messenger RNA levels, and protein loss in cell lines of affected individuals. Morpholino-mediated knockdown in a zebrafish model demonstrates that loss of the evolutionarily highly conserved CWF19L1, whose normal biological function is unknown, alters cerebellar morphology and causes movement abnormalities.
CONCLUSIONS: Our results suggest that CWF19L1 mutations may be a novel cause of recessive ataxia with developmental delay. Our research may help with diagnosis, especially in Turkey, identify causes of other ataxias, and may lead to novel therapies.

PMID: 25361784 [PubMed - indexed for MEDLINE] http://dlvr.it/8x1dyc

GABAA receptor biogenesis is impaired by the γ2 subunit febrile seizure-associated mutation, GABRG2(R177G).

PubMed:
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GABAA receptor biogenesis is impaired by the γ2 subunit febrile seizure-associated mutation, GABRG2(R177G).

Neurobiol Dis. 2014 Sep;69:215-24 

Authors: Todd E, Gurba KN, Botzolakis EJ, Stanic AK, Macdonald RL

Abstract
A missense mutation in the GABAA receptor γ2L subunit, R177G, was reported in a family with complex febrile seizures (FS). To gain insight into the mechanistic basis for these genetic seizures, we explored how the R177G mutation altered the properties of recombinant α1β2γ2L GABAA receptors expressed in HEK293T cells. Using a combination of electrophysiology, flow cytometry, and immunoblotting, we found that the R177G mutation decreased GABA-evoked whole-cell current amplitudes by decreasing cell surface expression of α1β2γ2L receptors. This loss of receptor surface expression resulted from endoplasmic reticulum (ER) retention of mutant γ2L(R177G) subunits, which unlike wild-type γ2L subunits, were degraded by ER-associated degradation (ERAD). Interestingly, when compared to the condition of homozygous γ2L(R177G) subunit expression, disproportionately low levels of γ2L(R177G) subunits reached the cell surface with heterozygous expression, indicating that wild-type γ2L subunits possessed a competitive advantage over mutant γ2L(R177G) subunits for receptor assembly and/or forward trafficking. Inhibiting protein synthesis with cycloheximide demonstrated that the R177G mutation primarily decreased the stability of an intracellular pool of unassembled γ2L subunits, suggesting that the mutant γ2L(R177G) subunits competed poorly with wild-type γ2L subunits due to impaired subunit folding and/or oligomerization. Molecular modeling confirmed that the R177G mutation could disrupt intrasubunit salt bridges, thereby destabilizing secondary and tertiary structure of γ2L(R177G) subunits. These findings support an emerging body of literature implicating defects in GABAA receptor biogenesis in the pathogenesis of genetic epilepsies (GEs) and FS.

PMID: 24874541 [PubMed - indexed for MEDLINE] http://dlvr.it/8yL4M7