If you're looking to excise a band from a PAGE gel after using Immunoprecipitation to purify a protein, staining all the protein in the gel can be useful. In steps the Coomassie Brilliant Blue dye. Originally developed for textiles, it's now routinely used to stain protein. There are many protocols out there for this process, but the one we find works best is below. It's also compatible with downstream mass spectrometry.
Installing the latest version of Ruby into Ubuntu 14.04 is painless using the method desribed here. If you're using bash that is! If you're using Z shell, which is what all the cool kids are using these days, you need to insert the Ruby environment paths into your Z shell environment instead.
Thawing frozen mononuclear cells ‘ficolled’ from a peripheral blood or bone marrow aspirate isn't as trivial as dealing with most cell lines. However, the following steps can help maximise the recovery of healthy cells.
- Thaw the cells quickly in a 37 °C waterbath
- Chill ~10 mL cold thawing buffer (IMDM, 10% FBS, 10 µg/mL DNAse) in a 15ml centrifuge tube
- Add thawed cells to this in a drop wise fashion with gentle mixing
- Rest for 10 minutes on ice
- Centrifuge at 200 × g for 5 minutes
- Discard supernatant being careful not disturb the pellet
- Resuspend cell pellet in desired volume (typically 1 mL) of buffer or complete media and count
For those who aren't familiar with endotoxins, QIAGEN provide a good primer on the subject and why it matters. Long story short, endotoxin contamination in plasmid preps is one of the biggest factors in getting mammalian cells to play nice with foreign DNA. A number of years ago I came across this paper by Ma et al 2012 and it became part of my routine plasmid prep. I saw improvements in viral titre from my packaging cells, improved nucleofection rates and better transfection.
"the same endotoxin removal efficiency when compared with the temperature transition extraction method."
The beauty in their method is its simplicity. The solutions to be prepared are simple and cheap and keep for a long time.
There are many suitable hosts for the multitude of plasmids used in a molecular biology lab.
- NEB Stable are a new clone from NEB. I have been using them for a while and they seem to be perfect for those plasmids that are likely to recombine, such as lentivirus constructs
- Stbl3 have been around longer, but are EndA+, so they tend to give a lower yield or partially degraded DNA