Lab safety No food, backpacks in lab No open-toed shoes Goggles Make sure you know the location of safety equipment Buddy system

Notebooks (p.4) No dictated format (bound?) Goals: –replicate the experiment Procedures Real-time modifications of procedures –understand outcomes Observations Raw data Interpretation of data

Notebooks Title Purpose Procedures –Calculations, etc. Observations –Raw data, processed data Conclusions

Lab reports (see due date on p. 1) Two “short” reports –Experimental procedures & Results sections Several sets of DAQs One “formal” report –JBC-style (Abstract, intro, exp proc, etc.) –Includes two due dates: rough draft & final Experimental presentation

Short reports (see p. 5) Title –Appropriate to the research being done Bad title: “Introduction to biochemical techniques”

Short reports Materials & Methods/Experimental Procedures –Terse, to-the-point –Someone else should be able to replicate the experiment –Concise vs. thorough –Passive tense

We obtained Bradford reagent from the refrigerator at the north end of room 207. We added 2.0 mL of Bradford reagent to plastic cuvettes. Paul added the protein samples containing Bovine serum albumin into the reagent. Dave inserted the cuvettes into the sample holder in the Cary UV/Vis spectrophotometer, and Mary pressed the button to read the absorbancy at 595 nm.

Aliquots (50  L) of bovine serum albumin (BSA) standards or the unknown sample were added to 2.0 mL Bradford reagent in plastic cuvettes. After a five minute incubation at room temperature, the absorbances at 595 nm were determined with a UV/Vis spectrophotometer (Varian, Inc.).

Short reports Results –Narrative (still passive tense) –More description of motivation –Assume a relatively naïve reader You yesterday

Short reports Difficulties –Determining what numbers go where ie. Making a standard curve: do you need to say in the M&M that you made standards of 0.05, 0.1, 0.2, 0.3, etc.  g/mL BSA? Results? Figures? –Concise vs. thorough –Organization Hint: use subtitles –Scientific language The final yield was pretty good. –Hint #2: Look at JBC articles!

Techniques Spectrophotometers Micropipettors

Pipettors Fragile/easy to break! Can be very accurate – can be very inaccurate

Pipettors Basic features –Disposable tip Cross-contamination –Dial Set volume –Plunger –Eject button (tip discard)

Pipettors Appropriate volumes –0.5 – 10  l (P10) – (P100) – (P1000) (1000  l = 1ml) – More accurate closer to upper limit –eg. use P100 for 100  L volumes

Pipettors How to choose volume How to seat tip Plunger ‘stops’ –Top –1 st push –2 nd push (expel)

Pipettors Watch tip –Air bubbles –Liquid on tip (drops carried over) –Liquid in tip (remaining after release) Dispensing –Into empty container: against the side –Into “full” container: immerse tip

Pipettors Sources of error –Tip not fully seated –Air bubble (not enough volume) –Drop on outside (too much volume) –Liquid remaining (not enough volume) –Too slow for time sensitive experiment?

Spectrophotometry

Light absorbed by a sample –Depends on: Concentration of absorbing species (how much) Path length (machine – constant) ‘Molar absorptivity’ (identity of absorbing species) A = εCL Final result Easily measurable (1 cm) Standard curve

Determining protein concentrations 0.5 mg/ml = 0.5  g/  l 0.5 mg/ml = 500  g/ml

Determining protein concentrations Direct: protein itself is the light-absorbing species –UV absorbance by peptide bond (amide) ~220 nm –UV absorbance by aromatic functional groups ~280 nm –Non destructive –Lower sensitivity –Interference by other compounds –Protein content (A280) eg. protein with zero Y, W amino acids will absorb little or no light at A280

Determining protein concentrations Indirect –Absorbance by environmentally- sensitive dye –Dye changes color when bound to protein –Higher sensitivity, reproducibility (Vis) –Cheap & easy –Destructive: sample no longer useable

Determining protein concentrations Bradford dye (Coomassie Brilliant Blue) –Red, no protein –Blue, protein-bound Which wavelength to use? Binds to positive charges –How universal is a standard curve?