recovered Protocols

Category: Genetics and Genomics: Cancer Genetics Protocols

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded tissue can be used. - [Read Clonality - X Chromosome Inactivation Assay Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for desalting of peptides and protein mixtures by RP-HPLC techniques. The RP-HPLC technique can be used to "desalt" peptide or protein samples derived from extraction procedures, from chemical reactions such as reductive alkylation in the presence of urea or guanidine hydrochloride, citraconylation, iodination, or cyanogen bromide cleavage, or recovered from other chromatographic separation. - [Read Desalting of Peptides and Protein Mixtures by RP-HPLC Techniques Protocol]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Bacteriophage {lambda} particles are recovered from bacterial lysates by precipitation with polyethylene glycol at high ionic strength. - [Read Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Cryopreserved PBMCs are a common specimen source for studies of immunological responses to vaccines, immunotherapies, etc. The health and viability of cells recovered post-cryopreservation are of course critical to the success and accuracy of immunological assays performed on them. This protocol standardizes PBMC isolation and cryopreservation techniques, specifically for the assessment of thawed cells by cytokine flow cytometry. - [Read Protocol for Isolation, Cryopreservation, and Thawing of PBMCs]

Category: Yeast Protocols: Yeast Protein Protocols

This assay directly measures the stability of a specific protein in yeast. Following pulse-labeling of yeast cells, the target protein is recovered by denaturing immunoprecipitation. - [Read Pulse-Chase Assay for Measuring Protein Stability in Yeast Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography. Fragments of DNA recovered from agarose gels are sometimes poor templates or substrates in subsequent enzymatic reactions.  This problem can be solved by binding the DNA to a positively charged matrix, such as DEAE-Sephadex or DEAE-Sephacel, in buffers of low ionic strength.  After washing the matrix, the DNA is eluted by raising the strength of the buffer. - [Read Purification of DNA Recovered Anion-exchange Chromatography Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

DNA fragments separated by electrophoresis through gels cast with low-melting-temperature agarose are recovered by melting the agarose and extracting the resulting solution with phenol:chloroform.  The protocol works best for DNA fragments ranging in size from 0.5 kb to 5 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

Chromatography on oligo(dT) columns is the preferred method for large-scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian cells. Typically, between 1% and 10% of the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA. Because the method can be frustratingly slow, it is not recommended for purification of poly(A)+ RNA from multiple samples. For this purpose, batch elution (Selection of Poly(A)+ RNA by Batch Chromatography) is the better choice. - [Read Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography - Subscription Required]