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 northend of room 207. We added 2.0 mL of Bradford reagent to plastic cuvettes. Paul added the protein samples containingBovine 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) standardsor the unknown sample were added to 2.0 mL Bradford reagent in plastic cuvettes. After a five minute incubationat 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 – 10l (P10)– 10-100 (P100)– 100-1000 (P1000) (1000 l = 1ml)– 1000-5000

• More accurate closer to upper limit– eg. use P100 for 100L volumes

Pipettors

• How to choose volume

• How to seat tip

• Plunger ‘stops’– Top– 1st push– 2nd 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

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)Standardcurve

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?