Presentation on theme: "Working in a BMB lab…. Some basic ground rules: 1)Know the theory behind everything you do and every kit that you use! If you don’t, you will end up protocol."— Presentation transcript:
Working in a BMB lab…. Some basic ground rules: 1)Know the theory behind everything you do and every kit that you use! If you don’t, you will end up protocol flip-flopping instead of troubleshooting. 2)Don’t 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 don’t necessarily offer advantages. 3)Consult with your colleagues. No need to reinvent the wheel. 4)Be vigilant about the effectiveness of your reagents.
Working in a BMB lab…. 4) Be vigilant about the effectiveness of your reagents.
Some random thoughts about proteins DNA is pretty tough, proteins are fairly robust. But, don’t 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.
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?
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 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) Many proteins can be lyophilyzed (freeze-dried) and stored at room temperature for years.
And, even more random thoughts…. Include appropriate protease inhibitors!
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)
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 protein:protein interactions Zwitterionic (amphoteric) detergents (CHAPS, zwitter- gent); can overcome protein:protein interaction while causing less protein denaturation
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.
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: Cu 2+ 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
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.
You want how much protein? Mass of protein to mole of protein: Protein Size 1 ug 1 nmol (Da, g/mol) 10,000100 pmol 10 ug 50,000 20 pmol 50 ug 100,000 10 pmol100 ug 150,000 6.7 pmol150 ug Note: relative gel staining intensity is size dependent.
Concentrating protein Smaller volumes (< 5 mL). Centrifugal filtration through spin-filters. These have MW pores that retain larger proteins. (5K -250K MWCO). Larger volumes (>20 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.
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.
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)
Web-based tools…. Variety of on-line resources available for the analysis of protein structure/function. Biospider (biospider.ca)biospider.ca FASTA (fasta.bioch.virginia.edu) The protein databank (www.rcsb.org)www.rcsb.org Nature Protocols (www.nature.com/nprot/index.html)www.nature.com/nprot/index.html EXPASY Expert Protein Analysis System (ca.expasy.org)