BIOC3570 F24 01 Protein Handling PDF

# A few important concepts ## Assay criteria - technical - Accuracy - how close is the measured value to the "true" value - Precision - given a constant amount of analyte, how similar are the readings produced by repeated measurements - Resolution - are the analyte peaks separated enough so that t...

# A few important concepts ## Assay criteria - technical - Accuracy - how close is the measured value to the "true" value - Precision - given a constant amount of analyte, how similar are the readings produced by repeated measurements - Resolution - are the analyte peaks separated enough so that their contributions can be measured without mutual interference - Sensitivity - what is the minimal amount of material you can reliably quantify - Specificity - does the assay measure only the targeted analyte? - Minimal signal from components other than the analyte (false positives) - Are all possible variants of the analyte reliably detected if present (false negatives) ## Accuracy and Precision The image shows a diagram of two bullseyes, representing accuracy and precision. - The top left shows targets scattered around the bullseye. This represents **inaccurate** and **imprecise** data. - The top right shows targets clustered close together but not in the centre. This represents **accurate** but **imprecise** data. - The bottom left shows targets clustered together in the centre but not in the bullseye. This represents **inaccurate** but **precise** data. - The bottom right shows targets clustered in the centre and in the bullseye. This represents **accurate** and **precise** data. ## Assay criteria - practical - Throughput/speed - can the assay be performed quickly with minimal effort - Multiplexing - can multiple analytes be analyzed in a single run, for example, on a plate reader (allows us to get a broader view of the sample) - Cost - How much does each sample analysis cost in terms of: - Reagents consumed - Sample consumed (potentially expensive) - Labour ## Be aware of experimental precision! - Always be aware of considerations of accuracy and precision - Here is a paper that reports protein yields to 6 or 7 significant figures - Better than 1 part per million, or 0.0001%. The measurement was made with the Lowry assay. Accurate to ~20%! ## Table 2. Purification of a trypsin inhibitor from chickpea seeds | Step | Total TIA (TUI) | Protein (mg) | Specific activity (TUI.mg-1) | Recovery (%) | Fold purification | |-------------------|------------------|---------------|----------------------------|---------------|--------------------| | Crude extract | 30391.90 | 13040.00 | 2.32 | 100.00 | 1.00 | | Heat denaturation | 26735.40 | 11626.00 | 2.29 | 88.00 | 1.01 | | NH4(SO4)2 ppt | 23664.00 | 6246.46 | 3.78 | 77.90 | 1.62 | | DEAE-Sephadex A-25 | 15787.00 | 217.63 | 72.54 | 51.94 | 31.26 | | Sephadex G-75 | 8876.00 | 63.00 | 140.88 | 29.20 | 60.46 | *Kansal et al., Braz. J. Plant Physiol. 20, 2008* ## Separations and Spectroscopy - These are the enabling technologies of modern biochemical analysis - They are especially powerful when used in combination - We will study many examples of both technologies, including: - Separations: Involve physical or chemical methods to divide a mixture - Binding-based: Chromatography - Physics-based: Electrophoresis and centrifugation - Spectroscopy: Measuring how substances will interact with electromagnetic radiation - UV-visible and fluorescence spectroscopy - Mass spectrometry - Our emphasis will be on the characterization of proteins # Proteins: Properties, handling, stability and reagents ## Protein solutions, protein problems # Proteins are highly organized amino acid polymers - Proteins are polymers built of an arbitrary, genetically encoded sequence of the 20 L-amino acids (plus possible covalent modifications and/or cofactors). - Most proteins fold into a compact, well-defined 3D structure. - The exceptions are intrinsically disordered proteins - these play specialized roles (protein interactions and signaling, stress protection). # The key amino acids in analytical biochemistry - Proteins are made from amino acids - Amino acids can be broadly divided into polar (Arg, Lys, His, Asp, Glu, Asn, Gln, Ser, Thr, Tyr) and non-polar (Trp, Phe, Met, Leu, Ile, Leu, Val, Pro, Ala, Gly, Cys). - There are eight amino acids that are especially important in analytical biochemistry: Arg, Lys, Glu, Asp, Trp, Tyr, Cys & His. - The residues are those that carry charges, are chemically reactive, are good metal binders, and/or absorb (and possibly fluoresce) light in the UV-visible range. - Make sure you know these eight and their properties. - For other amino acids, know at least polar vs non-polar. ## Table of Amino Acids | Polar Amino Acids (Three-letter) | no Acids (Three-letter / One-letter) | |----------------------------------|-----------------------------------| | Lysine / K (Lys) | (Trp) | | Histidine / H (His) | F (Phe) | | Aspartic Acid / D (Asp) | (Met) | | Glutamic Acid / E (Glu) | ) | | Asparagine / N (Asn) | ) | | Glutamine / Q (Gln) | ) | | Serine / S (Ser) | ) | | Threonine / T (Thr) | ) | | Tyrosine / Y (Tyr) | /S) | *The important 8* | Amino Acid (Three-letter / One-letter) | Polarity | Charge at Physiological pH | Side Chain Type | Hydrophobicity/Hydrophilicity | pKa of Side Chain | |--------------------------------------|----------|-----------------------------|-----------------|--------------------------------|----------------------| | Arginine (Arg / R) | | | | | | | Lysine (Lys / K) | | | | | | | Glutamic Acid (Glu / ) | | | | | | | Aspartic Acid (Asp / ) | | | | | | | Tryptophan (Trp / ) | | | | | | | Tyrosine (Tyr / Y) | | | | | | | Cysteine (Cys / C) | | | | | | | Histidine (His / H) | | | | | | *Lower the pKa, the more acidic you are.* # Negatively charged: Asp and Glu - Asp and Glu differ in that Asp has one linking methylene, Glu has two. This is why aspartate is more acidic (lower pKa). - The carboxylate groups are almost always negatively charged, but can be neutralized under strongly acidic conditions. # Positively charged: Arg and Lys - Arginine has a positively charged guanidine group. The guanidine group is only deprotonated at very strongly basic pH. It is essentially always positively charged. - Lysine amine group is positively charged. pKa is ~10.5. The amine group (also present on the N-terminus) is only deprotonated at strongly basic pH. # Positively charged (sometimes): Histidine - This histidine side chain contains imidazole ring. - This ring is protonated at slightly acidic pHs. - pKa ~ 6.5. If protonated, +1 charge; otherwise 0. - Histidine is good at binding metal ions, a property exploited in metal ion affinity purification. # Aromatic amino acids: Tyr and Trp - Tryptophan cannot be - It absorbs UV light strongly, and re-emits it strongly as fluorescence - Trp is the rarest amino acid. - The tyrosine OH group can be deprotonated, creating a phenolate (negatively charged) - Tyrosine (Tyr; Y) absorbs UV light less effectively than Trp and does not fluoresce. # Cysteine - Cys has a reactive thiol side chain - Cys can fairly readily be deprotonated - pKa is around 8.5; this gives Cys a negative charge - Cys's thiol group is the best nucleophile in a protein; it enables a wide variety of experiments that depend on specific chemical labelling - However, it's generally unreactive if it is buried in the protein's core - Cys can react with a second Cys to form a disulfide bond - Cystine absorbs UV light, albeit quite weakly - Cysteine is relatively rare in proteins # Side chains pKa values depend on the structural environment - Within a protein structure, nearby residues can stabilize one charge state of a residue over another. For example:&#x20; - A nearby positively charged residue will stabilize a negatively charged side chain - A non-polar environment will favour the neutral state - This alters that residue's pKa. - For example, in xylanase, adjacent residues destabilize a negative charge on Glu172. - Glu172 has a measured pKa of 6.8 (>>4.5) - The standard pKa values are average values, and specific residues in a given protein are often show pKa values shifted 1-2 pH units from this. # Amino acids differ in their natural abundances | AA | Frq | AA | Frq | |---|---|---|---| | A | 7.4 | L | 7.6 | | R | 4.2 | K | 7.2 | | N | 4.4 | M | 1.8 | | D | 5.9 | F | 4.0 | | C | 3.3 | P | 5.0 | | E | 5.8 | S | 8.1 | | Q | 3.7 | T | 6.2 | | G | 7.4 | W | 1.3 | | H | 2.9 | Y | 3.3 | | I | 3.8 | V | 6.8 | *Observed frequency in vertebrates (%)* - With 20 a.a., you might expect each to be ~5% of the average protein - However, some a.a. are relatively rare while others are relatively common - In particular, Cys, Tyr and His are somewhat rare, Trp is very rare. - Charged residues are slightly enriched. - With these average abundances and the MW of each amino acid, we can calculate that proteins on average weigh 110 Da per a.a. - So a 300 a.a. protein weighs ~33 kDa. # The organization of protein structure - Proteins bury non-polar amino acids in the core of the protein (orange spheres). - Polar residues cover most of the solvent exposed surface of protein (blue sticks). - Here they interact with water, helping solubilize the protein. ## This organization is generally critical for the protein's function # Water is critical to protein stability - Water molecules are bound by hydrogen bonds all over the surface of a protein molecule. - Some water is also deeply buried in the structure. - These water molecules stabilize exposed polar side chains, and solvate charged groups and ions - Most proteins cannot maintain their structures in the absence of water. - Proteins generally need to stay hydrated at all times. - Anything that disrupts normal water structure - dehydration, freezing, ## Structural waters in lysozyme (1HEW) - Inorganic solvents, high concentrations of salt - can potentially disrupt dissolved proteins # Protein structures are only marginally stable - Protein structure is stabilized by a significant number of weak interactions (van der Waals, hydrogen bonds etc.), and the hydrophobic effect. - Structured proteins are only marginally stable - the difference in being folded allows it to be dynamic. - This allows the protein to be dynamic enough to perform its function. - However, it also means that proteins can relatively easily become unfolded e.g. by the application of heat # Unfolding and aggregation inactivate proteins - Proteins that become partly unfolded have a certain innate ability to refold (smaller proteins generally do this more readily). - When proteins unfold, hydrophobic core residues become exposed. - These can stick to other unfolded proteins, forming aggregates that prevent refolding. - Aggregates can appear as (off-)white precipitate in the solution. - Aggregates can also be too small to be visible, but can still be detected e.g. by their light scattering properties. - Note - proteins can also precipitate by weak hydrophilic interactions while folded; such precipitates are generally reversible (e.g. salting out) # Proteins are sensitive to their chemical and physical environment - Chemical reagents are only inactivated by undergoing irreversible chemical reactions. - Proteins are more sensitive as they may also lose activity if they unfold, even if their chemical structure is unaltered. - Proteins have evolved to be stable in a specific environment - e.g. the cytosol of a cell, a particular multiprotein complex, a specific membrane, sea water... - Any significant environmental change can potentially destabilize the native structure of a protein and result in its (possibly permanent) inactivation. - This can include changes in temperature, pH or salt concentration, exposure to solvents, drying out, freezing... # Proteins are individuals that tolerate adversity - Some proteins are highly tolerant of a range of conditions, or will readily reactivate when returned to tolerable conditions. - Other proteins require very specific conditions, and/or are easily irreversibly inactivated. - Proteins are individuals, and working with any new protein involves getting familiar with its "likes" and "dislikes". - In general, most proteins are stable under conditions that resemble their natural environment. - But this can be hard to completely mimic... # Protein concentration The image shows a diagram of a cell, a test tube and a plate. This represents proteins in a cell, purified protein and a protein assay respectively. - Purified proteins for biochemical experiments are generally prepared at 0.1 - 50 mg/mL - Individual experiments (e.g. enzyme assays) may use very little protein, e.g. ~1 g/mL - However, total protein concentration in cells is generally ~400 mg/mL - This extreme dilution can destabilize proteins by reducing the entropic cost of unfolding (loss of crowding), or by dissociating complexes # "Soluble" protein solubility spans -5 orders of magnitude - The total protein concentration in the cytosol is around 400 mg/mC (so cytosol is ~1/3rd protein by volume) - Individual proteins are less soluble when pure. - Some proteins are soluble at > 100 mg/mL. - Other proteins precipitate at < 1 μ/mL. - Solubility is strongly influenced by buffer conditions - pH, salt concentration and type, other solutes, the presence of organic solvents... - So poorly-soluble proteins are made at least somewhat soluble by optimizing buffer conditions. # Excess dilution can dissociate complexes - Complexes will fall apart if diluted to concentrations around or below their effective dissociation concentration. - In addition, required co-factors can also be dissociated from proteins. - In either scenario, biological activity may be lost, possibly irreversibly. - Most long-term protein complexes are stabilized by sub-nanomolar interactions, and will maintain function at 1 nM concentration. - Some protein complexes associate more weakly, and can be prone to dissociate if the protein becomes too dilute. <start_of_image> え # Protein adsorption - Proteins have both polar and non-polar surfaces, and their surfaces tend to be "pre-organized" so binding occurs with minimal entropic cost. - Proteins can therefore potentially adhere to a wide variety of surfaces. - Biochemical equipment and disposables are generally made from materials that proteins find less "sticky" (e.g. glass, stainless steel, non-polar plastics, polysaccharides) . - However, individual proteins may still find any of these materials irresistibly sticky. - Detergents, increased salt concentrations, or carrier proteins (e.g. BSA) may all help reduce adherence - Protein adsorption is much more noticeable if protein was dilute to begin with. # Sepharose column The image shows a column chromatography apparatus.&#x20; # Air - Air is very non-polar (it cannot form any hydrogen bonds). - Proteins can unfold at the air-liquid interface (which is effectively a very hydrophobic surface). - An everyday example is beaten egg whites creating a stable foam as incorporated air denatures the egg proteins. - While air is unavoidable, avoid creating excess air-water surface by introducing bubbles (foam in biochemical experiments is generally a bad sign) - denature your protein. # Mechanical forces - Proteins can be denatured by very high pressures (>5k atm), but are generally quite resistant pressures seen in biochemical labs. - For example, French pressure cell presses disrupt cells at ~2.5k atm, but rarely adversely affect proteins. - Shear forces can result in some proteins unfolding. - For example, some proteins will denature when being drawn through a narrow needle. # Proteins and temperature - As temperatures increase, more solvent kinetic energy is available to disrupt stabilizing interactions - Increasing temperatures will therefore unfold proteins, generally irreversibly - Temperatures up to 37 C are tolerated by most proteins; tolerance for higher temperatures is protein dependent - Conversely, proteins that evolved at high temperatures may function sub-optimally or precipitate at low temperatures. - In the absence of contradictory evidence, it is safest to work with proteins cold (on ice), but not frozen. - Protein assays are generally conducted somewhere between 4 C (ice) and 37 C (human body temperature). # Freezing and thawing proteins - Freezing is a process distinct from cooling, and is generally especially damaging to proteins. - During freezing, ice crystals of pure water grow. - Freezing therefore concentrates solutes, including proteins, in the remaining liquid phase. - The resulting high salt/solute/protein concentrations and pH changes (as [H+] or [OH-] increases) can lead to protein aggregation and inactivation. - In addition, water expands as it freezes, which induces mechanical stresses. - Avoid repeated freeze-thaw cycles, as most proteins will lose at least some of their activity every time they are frozen. # Flash freezing - a common protein storage strategy - Rapid freezing of protein solutions prevents ice crystal growth. - Instead, water forms glass-like vitreous ice, incorporating impurities like proteins. - Ethanol + dry ice or liquid nitrogen baths are used to achieve rapid freezing of small volumes of sample. - Best practice is to freeze small "single use" samples - enough for one set of experiments. - Protein needed for the day is thawed on ice; any excess is discarded at the end of the day. # Freezing proteins: cryoprotectants - Solutes that reduce (rarely completely) damage during freezing are called "cryoprotectants". - Many poly-alcohols, including sugars can be good cryoprotectants. - In part this is because they are reasonably good mimics of water in forming hydrogen bonds, but suppress ice crystal growth. - Glycerol is very commonly used. - Ethylene glycol, and trehalose (a disaccharide) are also relatively common. - High concentrations of cryoprotectants can keep solutions liquid at temperatures well below 0 C. # Proteins can be very freezing sensitive: e.g. Diacylglycerol Kinase - Human diacylglycerol kinase is very sensitive to freezing. - Protein loses ~97% of activity (relative to unfrozen protein) upon flash freezing. - However, glycerol at 25% or 50% preserves ~ 1/2 of activity. - Glycerol can also help stabilize proteins even in the absence of freezing. - 10 - 50% glycerol is often added to protein reaction and storage buffers. # Lyophilization - In lyophilization, a frozen solution subjected to a vacuum. - Water sublimates (evaporates from the solid phase) leaving the solutes behind as a dry powder. - This process is used for proteins, nucleic acids, and small molecules. - Many proteins will retain their structure and function through drying (though conditions may need optimizing). - Once in this form a protein can be stable for years, especially if kept cold. - The protein can be 'reactivated' by adding water. - This process is commonly used for commercially sold enzymes, and in the drug industry. # pH affects protein solubility, stability and activity - The isoelectric point (pI) of a protein is the pH at which its net charge is 0. - Because charge-charge repulsion helps solubilize proteins, the pI is generally the pH at which the protein is least soluble. - At extremes of pH, proteins will unfold, precipitate, and be permanently inactivated. - In addition, enzymes activity require that key residues be appropriately protonated or deprotonated. - Optimal enzyme activity is generally quite pH sensitive. - Biochemists use buffers to stabilize the pH of solutions. <start_of_image> え # Calculating pH changes in a buffer as a function of amount of added acid - alpha = [A'] / ([A'] + [HA]) - Definition: alpha is the fraction of the total A that is in the conjugate base form - Henderson-Hasselbalch equation: pH = pKa + log ([A']/[HA]) - rearrangement: alpha = (10^(pH-pKa)) / (10^(pH-pKa)) + 1 - Michaelis equation: alpha = [A'] / ([A'] + [HA])&#x20; - The degree of dissociation (α) of an acidic (or basic) group can be calculated as a function of pH (i.e. -log[H+]). - This type of analysis is important to understanding both the fractional charge on a protein side chain, and buffering # Buffers and buffering capacity - A buffer is a solution containing both a weak acid and its conjugate base. - Added acid primarily converts the conjugate base into its acid form. - This minimizes the amount of added free acid, and therefore minimizes the corresponding change in pH. - However, eventually almost all of the conjugate base will be converted, and further additions of acid will cause large pH changes. - (Note - biochemists also use the term buffer for the solution a protein is dissolved in - pay attention to context) # Biological buffers - Buffers should be chosen so that their pKa is close to the target pH: - Remember: buffers have minimal effect more than 1 pH unit from their pKa. - Buffers for biochemistry should also be non-toxic, highly soluble, not absorb light in the UV-visible range, not bind metals, and be chemically unreactive. - Ideally, they also need to be chemically simple (i.e. cheap). - Most buffers are **temperature sensitive**, e.g. a Tris buffered solution will decrease pH by 0.5 units cooling from 20 C to 4 C. - Adjust the pH of buffers at their intended use temperature. # Examples of common biochemical buffers - MES: pKa 6.2 - TRIS: pKa ~8.1 - HEPES: pKa 7.5 - Bis TRIS: pKa ~6.5 - TAPS: pKa ~8.4 These buffers are also known as "Good's buffers." - Most biological buffers use amine (NR(1-3)) groups as the conjugate base - The pka of this group is shifted by the presence of nearby charge stabilizing groups (often hydroxyl or sulfonates: R-SO-) - Varying the chemical structure produces buffers with usefully different pKa's. In addition, some simple organic and inorganic ions including acetate, citrate, phosphate or bicarbonate are also used in some contexts. # Acid/base protein denaturation - As pH is lowered, any neutral His residues become protonated (pKa ~6.5). - Asp and Glu residues (pKa ~4.5) become protonated, neutralizing their charges. - The protein then has many positive charges (Arg, Lys & His), and few or no negative charges. - This results in charge-charge repulsion, destabilizing the protein. - At high pH, His & Lys (and eventually Arg) will be neutralized while Cys & Tyr (along with Asp and Glu) become negatively charged. - This again will result in charge-charge repulsion, destabilizing the protein. - In either case, extreme pH will tend to unfold and denature proteins. - In general, acid denaturation occurs around pH 2-5, base denaturation at pH > 10. - However, the exact pH at which a given protein begins to inactivate/unfold is protein specific. - Some proteins are very acid/base stable (e.g. stomach enzymes work at pH 2), others are very sensitive. # Salts help stabilize proteins - Salts - paired anions and cations - contribute ionic strength to solutions. - These ions associate with charged protein side chains, shielding them from each other and promoting protein solubility. - Cellular salt concentrations are maintained at ~150 mM salt (mostly NaCl). - Salt at this concentration is routinely added to protein solutions, as it helps stabilize most proteins. - Some proteins can become prone to precipitation if salt concentrations are too low. - High salt concentrations can also precipitate proteins. - However, it depends on the salt... # Hofmeister series | | | |:---------|:------------------------------------------------------------------------------------| | Kosmotropes: **stabilize & precipitate** | Chaotropes: **denature & dissolve** | | **Strengthens the hydrophobic effect** | | | NH4+ | K+ | | SO42- | HPO42- | | | acetate | | | Cl- | | | SCN- | - Hofmeister (1888) discovered that different salts differ markedly in the effect they have on proteins. - For example, sulfate salts tend to stabilize proteins, but also precipitate them (strengthens the hydrophobic effect). - Thiocyanate, in contrast, tends to denature proteins, but will also help dissolve them. - These differences arise from differences in the solvation energies of these ions that in turn alter the hydrophobic effect.&#x20; # Chaotropes and kosmotropes - Compounds which tend to decrease the hydrophobic effect and therefore push proteins to dissociate from another and also denature/unfold are known as chaotropes. - In addition to salts, many organic molecules (e.g. ethanol, butanol) also act as chaotropes. - Urea and guanidinium are especially effective. - Chaotropes can be used to unfold proteins, e.g. to stop an enzymatic reaction at the end of an assay. - Kosmotropes are compounds that increase the hydrophobic effect, and therefore encourage proteins to both fold and interact. # Proteins need to maintain appropriate disulfide bonds, and avoid wrong ones - Two cysteine residues in close proximity & in an oxidizing environment can oxidize to form a covalent bond - This is called a disulfide bond (or disulfide bridge) - The cytoplasm is reducing. - Disulfide bonds only form in extracellular proteins, or proteins within organelles e.g. ER. - Disulfide bonds can be critical for locking the tertiary structure of a protein. - Inappropriate disulfide bonds can inactivate or precipitate a protein. # Reducing agents reduce disulfide bonds - Reducing agents will reduce protein disulfide bonds while becoming themselves oxidized. - For example - mercaptoethanol (BME) - Reducing agents are routinely added to preparations of proteins from reducing environments (eg. the cytosol, nucleus, chloroplast). - However, they should generally be omitted if the protein is from an oxidizing environment (extracellular, interior of the ER, Golgi, or other organelles). え # Reducing agents in biochemistry - BME is cheap, but volatile and has a very strong odor (traces are added to natural gas to give it a noticeable scent). - Dithiothreitol has two thiol groups and is non-volatile: - Thiol reducing agents can react to form covalent adducts with protein, or react with molecular oxygen. - Phosphine reducing agents do not form new covalent bonds when oxidizing, and are safer for sensitive applications - e.g. Mass spectrometry. - For example, triscarboxyethyl phosphine (TCEP) # Detergents solubilize hydrophobic groups - Detergents combine a water-soluble head group and a non-polar tail in a single molecule. - Ionic detergents (with a charged head group) are generally "harsh" and tend to denature (unfold) proteins. - This is useful for some applications (e.g. electrophoresis) - Non-ionic detergents (or zwitterionic detergents) are less harsh, and less prone to denaturing proteins. - They are useful for solubilizing proteins that have extensive non-polar surfaces, especially membrane proteins (which are otherwise insoluble). - Membrane proteins can be quite sensitive to the exact detergent used, so it may be necessary to try several non-ionic detergents to find one that maintains activity. # Common detergents in biochemistry - Sodium dodecyl sulfate (SDS) - Triton X-100 # Biological challenges: proteases - All organisms contain proteases. - If these remain present as trace contaminants after purification, they will slowly cleave susceptible proteins. - High purity minimizes the chance of protease contamination: - Protease inhibitors can be added to inhibit any proteases present: - Protease inhibitors are available as mixes - e.g. chelating agents to inhibit metalloproteases, benzamidine to inhibit serine proteases - Some proteins are much more susceptible to proteolysis - proteases often target local disorder or specific amino acids/sequences. - Specific recombinant proteases are also used as reagents: - e.g. cleaving proteins into peptides for MS, removing affinity tags after purification # Biological challenges: microbes - Proteins (and nucleic acids) are an excellent source of nutrition! - Protein solutions, even if dilute and highly purified, contain everything bacteria need to grow. - It is important to keep biochemical solutions as sterile as possible. - For example, use single-use plasticware, sterile water sources for buffers etc. - Low temperatures also help slow bacterial growth (and proteases!). - Bacterial biofilms will eventually foul any equipment surface kept wet for more than a few days if not completely sterile. - 20% ethanol or methanol will kill bacteria, and is generally used to protect equipment that is stored wet (e.g. chromatography columns).