News & Highlights: A recent collabaration between YRC researcher John Yates and Martin W. Hetzer applied proteomics to examine protein turnover in cells of the rat central nervous system. They found that extremely long-lived proteins associated with chromatin and the nuclear pore complex did not turn over, potentially exposing these proteins to harmful metabolites and accumulation of damage over time. Read more about their findings in the journal Science to learn more. [Read Article]
News & Highlights: YRC Researchers Michael MacCoss and William Stafford Noble have published a new algorithm, dubbed Barista, for identifying proteins in complex biological mixtures. Instead of subdividing the task into separate peptide and protein identification tasks, Barista applies a machine learning approach to identify proteins from source spectra as a single optimization problem. Read their publications in Mol. Cell Proteomics to learn more. [Read Article]
News & Highlights: YRC researcher Stan Fields has used protein mass spectrometry to identify 870 unique sites of ubiquitin attachment on 438 different proteins in the budding yeast Saccharomyces cerevisiae. The analysis was based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. Read his paper in Proteomics to learn more. [Read Article]
News & Highlights: Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease characterized by premature aging. In their recent collaboration, YRC researcher John Yates and collaborator Juan Carlos Izpisua Belmonte found induced pluripotent stem cells from HGPS patients lacked molecular characteristics associated with the disease, which were restored upon differentiation. See their paper in Nature to learn more. [Read Article]
News & Highlights: YRC researcher Stan Fields used the model organism Saccharomyces cerevisiae to probe the effects of nutritionally acquired metabolites on statins, a cholesterol-lowering drug widely prescribed to prevent heart disease. He found that copper and zinc ions impair the effect of statins by upregulating genes related to sterol production. Please read his paper in Molecular BioSystems to learn more. [Read Article]
News & Highlights: YRC researchers David Baker and Stan Fields have developed new technology for examining how a protein's sequence affects its function. This new technology is large-scale and may be applied to many in vitro or in vivo protein assays, providing a general means for studying the functional consequences of protein variation. Please read their paper in Nature Methods to learn more. [Read Article]
News & Highlights: The YRC collaborated with Sue Biggins at the Fred Hutchinson Cancer Research Center in Seattle to examine centromeres, whose proper function is critical to prevent conditions associated with cancer and some birth defects. This work, performed in yeast, was recently published in Molecular Cell, where Dr. Biggins proposes a new pathway for the regulation of centromeric function. [Read Article]
News & Highlights: Multidimensional protein identification technology (MudPIT) developed by the YRC was used in a recent collaboration with David Drubin at the University of California, Berkeley, to examine the assembly of actin networks in yeast. In his recent paper in Current Biology, Dr. Drubin describes the nucleation and assembly of these large protein complexes, and how MudPIT was used to characterize their composition. [Read Article]

Fusing GFP to the Carboxy Terminus of Your Favorite Protein

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Design oligonucleotide primers for polymerase chain reaction (PCR)

The same primers used for the pFA6a plasmid can be used for pDH3 and pDH5. See Wach et al., Yeast Vol. 13: 1065–1075 (1997) for a discussion of primer selection.
We typically use ~60 mer PAGE-purified oligos with ~40 bp homology to the gene of interest.

Forward primer: (5' to 3'):

(~40 bp upstream of stop codon)-GGTCGACGGATCCCCGGG

Reverse primer: (5' to 3' bottom strand):

(~40 bp downstream of stop codon)-ATCGATGAATTCGAGCTCG

Amplify the integration cassette from the plasmid

PCR:

A polymerase with relatively high fidelity and high activity is necessary. The Roche Expand Long Template PCR system has worked well for us. The following protocol assumes use of the Expand system.
We find it helps to linearize the plasmid with NotI before using it as a template for PCR. Only a 15 minute digestion is necessary.
For a 50 µl of PCR reaction, make two solutions:

Solution 1:

Reagent Volume (µl)
75 ng/µl linearized plasmid 1
100 pmole/µl forward primer 0.5
100 pmole/µl reverse primer 0.5
2.5 mM dNTP ( 2.5 mM each ) 10
dH2O 13

Solution 2:

Reagent Volume (µl)
Buffer #3 (provided) 5
Expand polymerase mix 0.75
dH2O 19.25
Mix solutions well individually. Combine and mix well again.
Run using the following PCR conditions:
  • 94°C for 2 minutes
  • 92°C for 10 seconds
  • 50°C for 30 seconds
  • 68°C for 4 minutes
Repeat previous three steps 9 times. Then,
  • 92°C for 10 seconds
  • 50°C for 30 seconds
  • 68°C for 4 minutes + 20 seconds per cycle
Repeat previous three steps 18 times. Then,
  • 68°C for 7 minutes
  • 4°C indefinitely
Run 5 µL on gel to confirm PCR reaction.

Transform yeast with the cassette

  1. Grow a 10 ml culture of diploid cells to ~90 Klett units (mid-log phase). This is enough for two transformations. Scale up as necessary.
  2. Pellet cells at 4°C at 5,000 x g for 5 minutes. Decant supernatant.
  3. Wash cells with 5 ml of dH2O.
  4. Resuspend cells in 100 µl of 100 mM lithium acetate (LiOAc).
  5. Transfer to two Eppendorf tubes.
  6. During cell preparation, boil sheared salmon sperm carrier DNA for 5 min and then place on ice for at least 2 minutes.
  7. Pellet cells in microfuge for 15 sec. Decant supernatant.
  8. Add IN THIS ORDER to one tube:
    1. 240 µl of 50% PEG (mol. wt. 3350)
    2. 36 µl of 1.0 M LiOAc
    3. 25 µl of salmon sperm carrier DNA (10 mg/ml stock solution)
    4. 45 µl of cassette (from PCR reaction above)
  9. Mix. Place in 30°C rotator and leave for 45 minutes.
  10. Heat shock in 42°C water bath for 25 minutes.
  11. Pellet at 6,000 x g for 1 minute.
  12. Resuspend in 100 µl of dH2O
  13. Plate mix on 2 YPD plates and incubate at 30°C overnight.
  14. Replica plate to the appropriate selective medium (YPD G418 or SD -his) the following day.
  15. After 2 days at 30°C, pick large colonies and streak for single colonies on selective medium.
NOTE: If transformation efficiency is low, try doubling the amount of carrier DNA or replacing your stock. In our experience, the carrier DNA is crucial for efficient transformation.
Finally one should confirm the correct integration by PCR.

Plasmids & Protocols