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Rapid and Easy Gel Disruption by Passing Through Pipette Tip with Centrifugation

ABSTRACT: We have developed an Easy Disruption method for Gel using pipet Tips (EDGe-Tip), in which polyacrylamide gel is passed through pipette tips by centrifugation in a few minutes to crush it into appropriately sized fragments. The size of the disrupted gel could be controlled by several parameters such as the strength of the centrifugal force and the size of the pipette tip hole. In shotgun proteomics, GeLC/MS/MS analysis combining SDS-PAGE and LC/MS/MS requires dicing and transferring gels to microtubes by hand, which are essential but annoying steps. Thus, EDGe-Tip was applied to the GeLC/MS/MS procedure instead of those steps. Peptides could be extracted more efficiently and variation in protein quantification is lower with EDGe-Tip than with manual dicing. We believe that this simple and rapid method of disrupting polyacrylamide gels can be useful for GeLC/MS/MS analysis and applied for experiments to extract biomolecules from the gels.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD042573 | JPOST Repository | Fri May 31 00:00:00 BST 2024

REPOSITORIES: jPOST

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			Action	DRS
	220529HN01_Manual1mm250ng_1_2.raw	Raw
	220529HN02_Manual1mm250ng_2.raw	Raw
	220529HN03_Manual1mm250ng_3.raw	Raw
	220529HN04_3000G250ng_1.raw	Raw
	220529HN05_3000G250ng_2.raw	Raw

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Project description:This study describes the application of the LongSAGE methodology to study the gene expression profile in Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. LongSAGE revealed the gene expression profile in promastigotes of L. i. chagasi. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle. The I-SAGETM Long kit (Invitrogen) was used to construct the L. i. chagasi library according to the manufacturer's recommendations. The following were included among the main steps: polyadenylates mRNAs were captured by oligo (dT) linked to magnetic beads and used for cDNA synthesis. The cDNA was digested with 60 U of NlaIII (anchoring enzyme) and the 3' ends of the cDNAs were isolated using the beads. The resulting cDNA 3' was divided into two equal portions and connected to two LongSAGE adapters, A and B. The long tags were released by the enzyme MmeI and linked to form ~130 bp ditags. Dilutions of 1:40 of the product were amplified in 27 PCR cycles (300 reactions in total). The precipitated PCR products were separated on 12% polyacrylamide gel and the 130 bp bands were cut out and the precipitated DNA was digested with NlaIII. The digestion products were separated on 12% polyacrylamide gel and ~34 bp ditags were cut out and linked to form concatemers. The concatemers were separated on 6% polyacrylamide gel and the fractions of 250-500 and 500-800pb were isolated and cloned into pZErO-1 vector digested with SphI. Vectors with concatemers were cloned in E. coli DH10b by electroporation and the isolated recombinant vectors were used as template for sequencing in the ABI Prism 3100 (Applied Biosystems).

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Rapid and Easy Gel Disruption by Passing Through Pipette Tip with Centrifugation

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