Working in a BMB lab

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Working in a BMB lab.

• Some basic ground rules
• Know the theory behind everything you do and
  every kit that you use! If you dont, you will
  end up protocol flip-flopping instead of
  troubleshooting.
• Dont endlessly protocol shop. If you have a
  procedure that works, stick to it. It is easy to
  become infatuated with nifty new techniques and
  kits that dont necessarily offer advantages.
• Consult with your colleagues. No need to reinvent
  the wheel.
• Be vigilant about the effectiveness of your
  reagents.

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Working in a BMB lab.
4) Be vigilant about the effectiveness of your
reagents.
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Some random thoughts about proteins

• DNA is pretty tough, proteins are fairly robust.
  But, dont get too casual! Degradation is the
  fear that permeates protein work.
• Basic rules
• Ice ice baby! Always have a bucket of ice handy
  when you are doing any protein work and put tubes
  on ice immediately after removing them from
  freezers, centrifuges, etc.
• Spin cold! The rotors in bench-top
  micro-centrifuges can get mighty warm.

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More random thoughts.

• Know your protein! Each protein has its own
  personality and the properties of individual
  proteins can vary greatly.
• Is it a membrane protein (i.e. requires
  detergents?)
• Is it heat stable?
• Does it polymerize?
• Does it have disulfide bridges ( X-S-S-X)?
• Can it be repeatedly frozen and thawed?

Do you require enzymatic activity? Are there
cofactors required?
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Protein Stability.
I like my protein stirred not shaken, please!
Excessive vortexing can denature
proteins. Freeze/thaw storage Fridge (5C),
Freezer (-20C) and ultrafreezer (-80C).

• Antimicrobial agents required at 5C (Na Azide)
• Stabilizers such as glycerol, salt, sucrose, BSA
  may be required at -20C/-80C

Many proteins can be lyophilyzed (freeze-dried)
and stored at room temperature for years.
NOTE Some proteins are very sensitive to pH
alterations. Changing temperature can change pH.
What is the buffering agent? (e.g., TRIS, ?pH/?T
-0.035)
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And, even more random thoughts.

• Include appropriate protease inhibitors!

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Stabilizing phosphorylation status

• Include appropriate protein kinase and
  phosphatase inhibitors to maintain protein
  phosphorylation!

General kinase inhibitors SDS, EGTA, EDTA NaF,
staurosporine Specific/selective kinase
inhibitors are available. General phosphatase
inhibitors SDS, ?-glyce?o-phosphate, sodium
orthovanadate, pyrophosphate Specific/selective
phosphatase inhibitors PP1, PP2A
PP2B/calcineurin (okadaic acid, microcystin, etc)
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Detergents and Proteins.
Most important property of detergent is the
critical micelle concentration (CMC, the lowest
detergent concentration at which micelles
form). Detergents are most commonly used for the
extraction of membrane proteins.
Ionic detergents (SDS, LiDS, sodium cholate,
sodium deoxycholate) highly denaturing Non-ionic
detergents (Triton X-100, Triton X-114, Nonidet
P-40, Tween 20, Octylglucoside) less likely to
disrupt proteinprotein interactions Zwitterionic
(amphoteric) detergents (CHAPS, zwitter-gent)
can overcome proteinprotein interaction while
causing less protein denaturation
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More on detergents.
The method for detergent removal depends on the
protein, the detergent and other buffer
components. A high CMC permits rapid removal by
dialysis.
Possibilities for detergent removal!
Ionic detergent 1. Gel filtration on G25
Sephadex 2. Dialyze in presence of mixed-bed
exchanger Non-ionic detergent 1. Gel filtration
on G200 Sephadex 2. Dialyze against DOC, then
remove DOC. 3. Velocity sedimentation in sucrose
gradient 4. Bind protein to affinity or ion
exchange then wash.
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Do I have protein in this tube?
The nature of your protein sample will dictate
what assay to use. Buffer composition is also
important (e.g. Triton X-100 absorbs at A280 and
interferes with most dye binding
assays) Bradford Coomassie blue dye based. Fast,
sensitive, accurate. Detergent interference. BCA
Cu2 ions interact with peptide bonds. Fast,
sensitive, accurate. Interference with
detergents/organic solvents. Time dependent color
development. A280nm measures absorbance of
aromatic amino acids. Sample not destroyed. Not
as accurate. Protein must have aromatic residue.
Also consider A215nm
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Do I have protein in this tube?
Biuret Measures peptide bonds. Rapid but not
accurate at low protein. Less salt
interference Lowry (Folin-Ciocalteu) Similar to
BCA. Very sensitive. Detergent compatible.
Depends on presence of tyrosine residues.
You cannot directly compare the results of one
assay method with another. Working with the
relative protein determined by a method (e.g.
BSA gives a value about 2-fold greater than its
weight for the Bradford assay). A standard curve
must be run every time you perform the assay.
BSA, ovalbumin and IgG are commonly used.
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You want how much protein?
Mass of protein to mole of protein Protein
Size 1 ug 1 nmol (Da, g/mol)
10,000 100 pmol 10 ug 50,000 20 pmol
50 ug 100,000 10 pmol 100 ug 150,000 6.7
pmol 150 ug
Note relative gel staining intensity is size
dependent.
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Concentrating protein

• Smaller volumes (lt 5 mL). Centrifugal filtration
  through spin-filters. These have MW pores that
  retain larger proteins. (5K -250K MWCO).
• Larger volumes (gt20 mL). Concentration by
  dialysis on solid sucrose, polyethylene glycol or
  aquacide.
• Capture and elution from a chromatography support
• Protein can be acid (TCA) precipitated and then
  re-solubilized after removal of TCA with cold
  acetone. (sometimes requires carrier protein)
• NOTE Increasing protein during tissue
  extraction sometimes larger volumes of buffer
  will provide more efficient extraction.

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Protein chromatography.
Proteins (like DNA and RNA) are routinely
separated and isolated on solid supports/resins.
Many of these materials have been miniaturized to
maximize binding efficiency. The three main
types of supports/resins are gel-filtration,
ion-exchange and affinity chromatography.
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Protein chromatography.
Gel Filtration the separation on the basis of
molecular size the resin contains pores which
trap/retard smaller molecules (Sephadex,
Sephacryl, Sepharose) commonly used for
desalting Ion Exchange the separation based on
charge electrostatic interaction of protein with
resin (anionic, cationic or mixed bed) commonly
used in protein purifications Affinity
Chromatography the separation based on natural
(ligand/protein) binding sites protein is
specifically and reversibly adsorbed by
immobilized ligand (glutathione-Sepharose,
ATP-Sepharose, biotin-agarose, protein A-agarose,
etc)
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Web-based tools.

• Variety of on-line resources available for the
  analysis of protein structure/function.
• Biospider (biospider.ca)
• FASTA (fasta.bioch.virginia.edu)
• The protein databank (www.rcsb.org)
• Nature Protocols (www.nature.com/nprot/index.html
  )
• EXPASY Expert Protein Analysis System
  (ca.expasy.org)