LB medium bottle and LB agar plate

Lysogeny broth (LB), a nutritionally rich medium, is primarily used for the growth of bacteria. It is also known as Luria broth or Luria-Bertani broth.

LB media formulations have been an industry standard for the cultivation of Escherichia coli as far back as the 1950s. These media have been widely used in molecular microbiology applications for the preparation of plasmid DNA and recombinant proteins. It continues to be one of the most common media used for maintaining and cultivating recombinant strains of Escherichia coli.

There are several common formulations of LB. Although they are different, they generally share a somewhat similar composition of ingredients used to promote growth, including the following:

• Peptides and casein peptones
• Vitamins (including B vitamins)
• Trace elements (e.g. nitrogen, sulfur, magnesium)
• Minerals

Peptides and peptones are provided by tryptone. Vitamins and certain trace elements are provided by yeast extract. Sodium ions for transport and osmotic balance are provided by sodium chloride. Bacto-tryptone is used to provide essential amino acids to the growing bacteria, while the bacto-yeast extract is used to provide a plethora of organic compounds helpful for bacterial growth.

Formulas

The formulations generally differ in the amount of sodium chloride, thus providing selection of the appropriate osmotic conditions for the particular bacterial strain and desired culture conditions. The low salt formulations, Lennox and Luria, are ideal for cultures requiring salt-sensitive antibiotics.

• LB-Miller (10 g/l NaCl)
• LB-Lennox (5 g/l NaCl)
• LB-Luria (0.5 g/l NaCl)

Misnomers

LB is also known as:

• Luria-Bertani broth (though this name is very widely used)
• Luria broth
• Lennox broth

The recipe for LB was formulated by Giuseppe Bertani and published in 1951. Over the years the acronym has been widely misconstrued. In the Postscript to his 2004 paper, “Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems”, Giuseppe Bertani clarified the original meaning of the acronym:

“My first paper on lysogeny, describing the modified single-burst experiment and the isolation of P1, P2, and P3, also contained the formula of the LB medium which I had concocted in order to optimize Shigella growth and plaque formation. Its use has since become very popular. The acronym has been variously interpreted, perhaps flatteringly, but incorrectly, as Luria broth, Lennox broth, or Luria Bertani medium. For the historical record, the abbreviation LB was intended to stand for “lysogeny broth.” (5, page 598).

Preparation

The following is a common method for the preparation of 1 litre of LB:

• Measure out the following:
  • 10g tryptone
  • 5g yeast extract
  • 10g NaCl
• Suspend the solids in ~800ml of distilled or deionized water.
• Add further distilled or deionized water, in a measuring cylinder to ensure accuracy, to make a total of 1 litre.
• Autoclave at 121°C.
• After cooling, swirl the flask to ensure mixing, and the LB is ready for use. Try your very best to keep it sterile!

Adjusting the pH

Prior to autoclaving, some labs adjust the pH of LB to 7.5 or 8 with sodium hydroxide. The downside of using sodium hydroxide is that the pH will not be buffered which means that the bacteria will rapidly change the pH as they grow. To get around this some labs prefer to adjust the pH with 5-10 mmol/L TRIS buffer, diluted from 1 mol/l TRIS stock at the desired pH. However, it is not absolutely necessary to adjust the pH for most situations.

Since the buffering with Tris will also be largely ineffective in the face of substantial bacterial growth, adjusting the pH of LB in this particular manner is usually unnecessary. As such, use of Tris in some broth recipes (especially when the culture will be stored at room temperature conditions for extended periods of time) may be considered a superstitious procedure without much scientific merit.

References

• Anderson, E. H. (1946). Growth requirement of virus-resistant mutants of Escherichia coli strain B. Proc. Natl. Acad. Sci. USA 32:120-128. PMID 16588724
• Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62:293-300. PMID 14888646 PDF
• Luria, S. E., and J. W. Burrous. (1957). Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74:461-476. PMID 13475269 PDF
• Lennox, E. S. (1955). Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1:190-206. PMID 13267987
• Luria, S. E., J. N. Adams, and R. C. Ting. (1960). Transduction of lactose-utilizing ability among strain of E. coli and S. dysenteriae and the properties of the transducing phage particles. Virology. 12:348-390. PMID 13764402
• Miller, J. H. (1972). Experiment in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
• Sambrook, J., E. F. Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
• Bertani, G. (2004). Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J. Bacteriology. 186:595-600. PMID 14729683 doi:10.1128/JB.186.3.595-600.2004

A pipette (also called a pipet, pipettor or chemical dropper) is a laboratory instrument used to transport a measured volume of liquid.

Use and variations

Pipettes are commonly used in chemistry and molecular biology research as well as medical tests. Pipettes come in several designs for various purposes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes. A pipette works by creating a vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw up and dispense liquid.

Pipettes that dispense between 1 and 1000 ?l are termed micropipettes, while macropipettes dispense a greater volume of liquid.

Glass pipettes

The original pipette is made of glass. It is more commonly used in chemistry, with aqueous solutions. There are two types. One type, the volumetric pipette, has a large bulb, and is calibrated for a single volume. Typical volumes are 10, 25, and 50 mL. Alternatively, Mohr pipettes are straight-walled, and graduated for different volumes such as 5 mL in 0.5 mL increments. The single volume pipette is usually more accurate, with an error of ± 0.1 or 0.2 mL.

The pipette is filled by dipping the tip in the volume to be measured, and drawing up the liquid with a pipette filler past the inscribed mark. The volume is then set by releasing the vacuum using the pipette filler or a damp finger. While moving the pipette to the receiving vessel, care must be taken not to shake the pipette because the column of fluid may “bounce”.

Piston-driven air displacement pipettes

Biohit Pipette

These are the most accurate and precise pipettes. They are more commonly used in biology, though they are commonly used by chemists as well. The plastic pipette tips are designed for aqueous solutions, and are not recommended for use with organic solvents which may dissolve the plastic.

These pipettes operate by piston-driven air displacement. A vacuum is generated by the vertical travel of a metal or ceramic piston within an airtight sleeve. As the piston moves upward, driven by the depression of the plunger, a vacuum is created in the space left vacant by the piston. Air from the tip rises to fill the space left vacant, and the tip air is then replaced by the liquid, which is drawn up into the tip and thus available for transport and dispensing elsewhere.

Sterile technique prevents liquid from coming into contact with the pipette. Instead, the liquid is drawn into and dispensed from a disposable pipette tip which is changed between transfers. Depressing the tip ejector button removes the tip, which is cast off without being handled by the operator and disposed of safely in an appropriate container.

The plunger is depressed to both draw up and dispense the liquid. Normal operation consists of depressing the plunger button to the first stop while the pipette is held in the air. The tip is then submerged in the liquid to be transported and the plunger is released in a slow and even manner. This draws the liquid up into the tip. The instrument is then moved to the desired dispensing location. The plunger is again depressed to the first stop, and then to the second stop, or ‘blowout’, position. This action will fully evacuate the tip and dispense the liquid. In an adjustable pipette, the volume of liquid contained in the tip is variable; it can be changed via a dial or other mechanism, depending on the model. Some pipettes include a small window which displays the currently selected volume.

Certain considerations should be observed to ensure maximum accuracy and repeatability:

• Operator consistency is paramount to repeatable operation. The necessity of operator practice and development of good pipetting practices and habits is absolute. Light guided pipetting aides are used to help reduce errors and speed up liquid handling protocols.
• When drawing up liquid the tip should be dipped 3 to 5 mm below the surface of the liquid, always at a 90 degree angle.
• When dispensing the pipette should be held at a 45 degree angle, and the tip placed against the side of the receiving vessel. Glass vessels are preferred; the surface tension of the glass provides additional torsion that results in complete evacuation of the tip.
• The tip must never be wiped off or blotted in any way, even from the exterior, while liquid is in the tip. These actions tend to attract and thus bleed off some of the liquid, resulting in decreased accuracy and repeatability.
• A dry tip should always be pre-wetted by drawing up and dispensing the chosen volume a minimum of three times. This action reduces the surface tension on the inside walls of the tip and also provides the proper level of inter-tip humidity, which reduces evaporation of the sample liquid.
• Most pipettes are calibrated “to deliver” (TD) and not “to contain” (TC). If they are TD pipettes they should not be rinsed after they have delivered their contents. If the pipette were calibrated TC it should be rinsed to obtain the correct amount of material. If the fluid to be measured is quite viscous or sticky (such as glycerol solutions) the pipette must be calibrated and in this case the outside of the tip must be carefully wiped with a lint free tissue to remove the adhering liquid – while being careful to not touch the opening of the pipette tip, which may require some practice. Accuracy in delivering liquids with high or low viscosity may require a “positive displacement” pipettor, which is quite distinct from an air displacement pipettor.
• For maximum accuracy, and especially necessary when calibrating the pipette, relative humidity in the ambient environment should be maintained between 50% and 75%, and in no case should the humidity be allowed to dip below 50%. This limits the rate of sample evaporation which can cause significant errors, especially at lower volumes.

The importance of operator skill cannot be overstated. A high-quality, well-calibrated pipette in the hand of an uninterested or untrained operator is an unreliable instrument. Additionally, there are four factors that can reduce the accuracy and repeatability of even highly-skilled operators, and these factors must be counteracted if optimal accuracy is to be achieved:

• Heat from the operator’s hand is absorbed through the handle of the instrument and transferred to the metallic components inside. If the pipette is operated continuously for a prolonged period of time this heat buildup becomes significant, causing the internal components to expand and changing the interplay between components. This reduces the consistency, accuracy, and repeatability of the instrument. The volume dispensed is dependent on the sizes of the piston and the springs that cause its travel. As these change in size the volume dispensed changes also. This effect is more pronounced in low-volume instruments. Additionally, the expansion of a metallic component that interacts with a non-metallic one that does not expand as readily in the presence of heat may cause the instrument to seem to stick, hang up, or react more slowly. Pipettes with thin handles are particularly susceptible to this phenomenon. Plumper handles are both more ergonomic and less likely to suffer from heat transfer problems. The best technique for maximum accuracy is to employ multiple pipettes and rotate them often, storing them between uses in a stand that holds them vertically.

• Operator fatigue is an often-overlooked but crucial component when seeking maximal accuracy and repeatability. Human beings are not robots, and repetitive motions cause stress in human joints and muscles. Even a well-trained and experienced operator will see a decrease in accuracy and repeatability as length of time on the job increases. It is for this reason that pipette calibration service providers that are dedicated to excellence limit the number of pipettes that can be calibrated by an individual technician to a maximum daily number. Each pipette, and each customer, deserves a high level of care in the treatment of the instrument. Additionally, some dedicated professionals train themselves to pipette ambidextrously, allowing them to reduce arm and finger strain by alternating hands. Another solution is choosing an electronic pipettor which significantly reduces hand fatigue. Once the operating button is touched the pipettor operates always the same way producing user independent accuracy and precision.

• Long-term pipette operation can lead to repetitive strain injuries (RSI), such as carpal tunnel syndrome. These disorders may cause significant reductions in accuracy and repeatability by altering the proper pipetting techniques that are crucial to achieving optimal accuracy. Preventive measures include learning to pipette with both hands and alternating their usage, taking frequent breaks while pipetting, and choosing the most ergonomic pipette available. Instruments with plumper handles are generally superior in this regard. On the other hand, electronic pipettors which operate with a light touch reduce RSI significantly.

• Letting the pipette “rest” for at least one minute after a volume change is made. This does not apply to single-volume instruments, also called set volume or fixed volume pipettes. A change in the dispensed volume of an adjustable pipette involves modifying the internal tensioning of a spring that governs the piston’s travel distance. Springs subjected to changing tensioning behave more smoothly and consistently when they are allowed to enjoy an interval of rest to settle into their new configuration. A pipette that is left idle for at least one minute after a volume adjustment will perform more accurately than one that is pressed into service prematurely. This is especially important when calibrating a pipette.

Calibration

For sustained accuracy and consistent and repeatable operation, pipettes should be calibrated at periodic intervals. These intervals vary depending on several factors:

• The skill and training of the operators. Skilled operators tend to operate the instrument more correctly and make fewer accuracy-robbing mistakes.
• The liquid dispensed by the pipette. Corrosive and volatile liquids tend to emit vapors which ascend into the pipette shaft even under proper operating conditions and may corrode the metal piston and springs, or the seals and o-rings that provide an air-tight seal between the piston and the surrounding sleeve.
• Proper and careful handling. Pipettes that are frequently dropped, are subjected to careless handling or horseplay, or that are not properly stored in a vertical position, will tend to degrade in accuracy over time.
• The accuracy required by the instrument. Applications requiring maximum accuracy also demand more frequent calibration. Instruments used for purely research applications or in educational settings generally require less frequent calibration.

Under average conditions, most pipettes can be calibrated semi-annually (every six months) and provide satisfactory performance. Institutions that are regulated by the Food and Drug Administration‘s GMP/GLP regulations generally benefit from quarterly calibration, or every three months. Critical applications may require monthly service, while research and educational institutions may need only annual service. These are general guidelines and any decision on the appropriate calibration interval should be made carefully and include considerations of the pipette in question (some are more reliable than others), the conditions under which the pipette is used, and the operators who use it.

Calibration is generally accomplished through means of gravimetric analysis. This entails dispensing samples of distilled water into a receiving vessel perched atop a precision analytical balance. The density of water is a well-known constant, and thus the mass of the dispensed sample provides an accurate indication of the volume dispensed. Relative humidity, ambient temperature, and barometric pressure are factors in the accuracy of the measurement, and are usually combined in a complex formula and computed as the Z-factor. This Z-factor is then used to modify the raw mass data output of the balance and provide an adjusted and more accurate measurement.

The colormetric method uses precise concentrations of colored water to affect the measurement and determine the volume dispensed. A spectrophotomer is used to measure the color difference before and after aspiration of the sample, providing a very accurate reading. This method is more expensive than the more common gravimetric method, given the cost of the colored reagents, and is recommended when optimal accuracy is required. It is also recommended for extremely low-volume pipette calibration, in the 2 microliter range, because the inherent uncertainties of the gravimetic method, performed with standard laboratory balances, becomes excessive. Properly calibrated microbalances, capable of reading in the range of micrograms (10^-6 g) can also be used effectively for gravimetric analysis of low-volume micropipettes.

Other pipette types

• Pasteur pipettes, also known as droppers are used to transfer small amounts of liquids, but are not graduated. Pasteur pipettes are made of plastic or glass.
• Transfer pipettes, are similar to Pasteur pipettes. However, they are made exclusively from plastic and their bulb can serve as the liquid-holding chamber.
• Serological pipettes are measuring pipettes that have graduations extending all the way to the tip.
• Mohr pipettes are measuring pipettes that resemble serological pipettes, with the primary difference that the graduations do not extend all the way to the tip.
• Dispensable pipettes are often made of plastic and intended to be used to administer medicine into the eye or ear of a patient (see image).

Pipette accessories

• Pipette fillers are used to fill the pipette easily, avoiding the need for mouth pipetting.
• Pipette helpers are battery-operated and are designed to be used with disposable pipette tubes. These pipettes cannot be calibrated and their accuracy is determined by that of the printed graduations on the disposable tubes.
• Light-guided pipetting systems are pipetting accessories which are computer based. They utilize flat screen LCD monitors or LED arrays to light up source and destination wells in microplates or vials for accurate well to well pipetting. Some of these systems use text to speech to alert the operator during plate or volume changes when pipetting lab protocols.
• Pipette tips. The pipettors and injection molded plastic disposable tips form together a reliable pipetting system. It is recommended to use original manufacturers tips to guarantee the precision and accuracy of the pipettes. The precision-made pipettor tips provide excellent reproducibility and accuracy. Pipettor tips are available in autoclavable boxes, refills and bulk packaging. Non-sterile, pre-sterilized and filtered tips are usually available in single trays as RNase, DNase and endotoxin certified free.

The smallest pipette

A zeptoliter pipette has been developed at Brookhaven National Laboratory. The pipette is made of a carbon shell, within which is an alloy of gold-germanium alloy. The pipette was used to learn about how crystallization takes place.^[1]

References

Although chlorine bleach is commonly employed in many laboratories as a neutralization procedure, it is not effective in removing the mutagenic properties of DAB. A potassium permanganate-sulfuric acid procedure is, however, an effective way of neutralizing this toxic compound.

1. Take up bulk quantities of diaminobenzidine tetrahydrochloride dehydrate in water and bulk quantities of the free base in 0.1 M hydrochloric acid so that the concentration of DAB does not exceed 0.9 mg/ml. Dilute solutions with the same buffer, if necessary, so that the concentration does not exceed 0.9 mg/ml.
2. For each 10 ml of solution, add 5 ml of 0.2 M potassium permanganate solution (31.6 g KMnO4 per liter of solution with water) and 5 ml of 2 M sulfuric acid solution (112 ml concentrated H2S04 per liter of solution with water).
3. Allow the mixture to stand for at least 10 hours.
4. Test to ensure pH of solution is between 6-9. Dispose solution down the drain with copious amounts of water.

Description

Lysogeny broth (LB), more commonly called Luria Broth,  agar plates are typically used as a growth substrate for the culture of bacteria (e.g., E. Coli). Selective growth compounds may also be added to the media, such as antibiotics. Individual microorganisms placed on the plate will grow into individual colonies, each a clone genetically identical to the individual ancestor organism (except for the low, unavoidable rate of mutation). Thus, the plate can be used either to estimate the concentration of organisms in a liquid culture or a suitable dilution of that culture, using a colony counter, or to generate genetically pure cultures from a mixed culture of genetically different organisms, using a technique known as streaking. In this technique, a drop of the culture on the end of a thin, sterile loop of wire is “streaked” across the surface of the agar leaving organisms behind, a higher number at the beginning of the streak and a lower number at the end. At some point during a successful “streak”, the number of organisms deposited will be such that distinct individual colonies will grow in that area which may be removed for further culturing, using another sterile loop.

Procedure

This recipie makes about 1 L of media, sufficient for 30 plates. Preparation time is approximately 2 hours.

1. To a flask of volume at least 2 L, add:

• 10 g Tryptone
• 5 g Yeast Extract
• 5 g NaCl
• 800 mL of distilled water

2. Stir the solution until everything is completely dissolved.
3. Add 400ul of 5N NaOH with stirring to adjust the pH.
4. Bring the liquid level up to to 1000 ml with distilled water.
5. Add 15g of granulated agar to the liquid and stir until the agar is dissolved (about 1 minute).
6. Remove the stir bar, cover the flask with aluminum foil and autoclave for 20 min using the liquid cycle.
7. Cool down the medium until it is cool enough to be held in the hands (about 40^oC).
8. While the media is cooling, spray and wipe the bench with 95% ethanol.
9. Open a bag of sterile 3″ empty plates and place them in stacks of 10 plates with the lids up. Save the bag for later storage of the plates.
10. Label the plates for proper identification:

• LB only – single vertical black band
• LB + Ampicillin – (optional black band) single vertical red band
• LB + Chloramphenicol – single vertical blue band
• LB + Kanamycin – (optional black band) single vertical green band

11. When the media has cooled, add the appropriate amount of antibiotic(s) to the medium and gently swirl to mix:

• 100ug/mL Ampicillin
• 34ug/mL Chloramphenicol
• 10ug/mL Kanamycin

12. At this point, you can pour the LB agar from the flask into a sterile 500-mL beaker for easier transfer onto the plates.
13. Sterilize the flask mouth by flame. If any bubbles are present in the agar, you can burst them passing the flame quickly over the LB agar solution.
14. Open the lid of the top plate and flame the beaker mouth, then pour the LB agar onto the plate until about half-way full.
15. The plates should stand at room temperature for a day before being bagged and stored. They may be used for experiments later the same day if required.
16. Store the plates upside down inside the bag, to prevent them from drying out, and store at 4^oC.

Loading controls are commonly used in gel electrophoresis techniques, such as western blotting, to verify that the gel lanes have been evenly loaded with sample material, and they are typically used to standardize the results from these studies. Since the proteins in loading controls are abundantly expressed, they also allow investigators to determine if there is an even transfer across the entire gel. A loading control is absolutely required when western blots are prepared for publication.

Beta Actin
Origin: Whole Cell / cytoplasmic
Molecular weight (kD): 43
Beta actin is one of six isoforms of actin. Actin is a globular, roughly 42-kDa protein found in all eukaryotic cells (except for nematode sperm) where it may be present at concentrations of over 100 ?M. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans. It is the monomeric subunit of microfilaments, one of the three major components of the cytoskeleton, and of thin filaments, which are part of the contractile apparatus in muscle cells. Thus, actin participates in many important cellular functions, including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. This loading control is not suitable for skeletal muscle samples. Changes in cell-growth conditions and interactions with extracellular matrix components may alter actin protein synthesis (Farmer et al, 1983).

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Origin: Whole Cell / cytoplasmic
Molecular weight (kD):30-40
Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for RT-PCR. However, many researchers report different regulation of GAPDH under specific conditions. Consequently, the use of GAPDH as loading control has to be carefully evaluated.

Tubulin
Whole Cell / cytoplasmic
Molecular weight (kD):55
Tubulin is one of several members of a small family of globular proteins. The most common members of the tubulin family are ?-tubulin and ?-tubulin, the proteins that make up microtubules. Each has a molecular weight of approximately 55 kiloDaltons. Microtubules are assembled from dimers of ?- and ?-tubulin. These subunits are slightly acidic with an isoelectric point between 5.2 and 5.8. Tubulin expression may vary according to resistance to antimicrobial and antimitotic drugs (Sangrajrang S. et al, 1998, Prasad V et al, 2000)

VDCA1/Porin
Mitochondrial
Molecular weight (kD): 31
Porins are beta barrel proteins that cross a cellular membrane and act as a pore through which molecules can diffuse. Unlike other membrane transport proteins, porins are large enough to allow passive diffusion – i.e., they act as channels that are specific to different types of molecules. They are present in the outer membrane of Gram-negative bacteria, the mitochondria, and the chloroplast.

Cytochrome C Oxidase (COXIV )
Mitochondrial
Molecular weight (kD):16
Cytochrome C Oxidase (COXIV) is located in the inner mitochondrial membrane and serves as the terminal enzyme complex of the mitochondrial electron transport chain. COXIV collects electrons that are transferred from reduced cytochrome C and donates them to molecular oxygen, resulting in reduction to water. COXIV is expressed at a consistently high level and thus makes a good mitochondrial loading control; however, many proteins run at the same size.

Lamin B1
Origin: Nuclear envelope
Molecular weight (kD): 66
The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane. The lamin family of proteins make up the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are thought to be involved in nuclear stability, chromatin structure and gene expression. Vertebrate lamins consist of two types, A and B. This gene encodes one of the two B type proteins, B1. This protein is not suitable for samples where the nuclear envelope has been removed.

TATA binding protein TBP
Origin: Nuclear
Molecular weight (kD):38
The TATA binding protein (TBP) is a transcription factor that binds specifically to a DNA sequence called the TATA box. This DNA sequence is found about 25-30 base pairs upstream of the transcription start site in some eukaryotic gene promoters. TBP, along with a variety of TBP-associated factors, make up the TFIID, a general transcription factor that in turn makes up part of the RNA polymerase II preinitiation complex. As one of the few proteins in the preinitation complex that binds DNA in a sequence-specific manner, it helps position RNA polymerase II over the transcription start site of the gene. However, it is estimated that only 10-20% of human promoters have TATA boxes. Therefore, TBP is probably not the only protein involved in positioning RNA polymerase II. This protein is not suitable for samples where the nuclear envelope has been removed.

Description

In molecular biology, c-Fos is a cellular proto-oncogene belonging to the immediate early gene family of transcription factors. c-Fos has a leucine-zipper DNA binding domain, and a transactivation domain at the C-terminus. Transcription of c-Fos is upregulated in response to many extracellular signals, e.g. growth factors. Additionally, phosphorylation by MAPK, PKA, PKC or cdc2 alters the activity and stability of c-Fos. Members of the Fos family dimerise with Jun to form the AP-1 transcription factor, which upregulates transcription of a diverse range of genes involved in everything from proliferation and differentiation to defense against invasion and cell damage.

The AP-1 complex has been implicated in transformation and progression of cancer, and both Fos and Jun were first discovered in rat fibroblasts.

The viral homologue of c-Fos, v-Fos, is found in the retrovirus Finkel-Biskis-Jinkins murine osteogenic sarcoma virus. In neuroscience research, neuroscientists measure expression of c-fos as an indirect marker of neuronal activity because c-fos is often expressed when neurons fire action potentials.

Staining procedure

1. This is a free-floating staining procedure for formalin-fixed brain tissue. Sections should be cut between 15-30 µm.
2. Transfer sections in 6-well plates loaded with PBS 0.1 M (one brain per well).
3. Rinse sections twice, 10 minutes each rinse, with PBS 0.1 M on a shaker.
4. Incubate sections with fresh 0.3% H₂O₂ in PBS 0.1 M for 30 minutes at room temperature on a shaker.
5. Rinse sections 3 x 10 minutes with PBS 0.1 M on a shaker.
6. Incubate sections with blocking solution  for 60 min at room temperature on a shaker.
7. Incubate sections with primary antibody diluted in blocking solution overnight at room temperature on a shaker.  With certain antibodies, to reduce background staining, consider an incubation for 2-3 days at 4°C.
8. Rinse sections 4 x 10 minutes with PBS 0.1 M on a shaker.
9. Incubate sections with biotinylated secondary antibody, diluted in blocking solution for 2 hours at room temperature on a shaker.
10. Rinse sections 4 x 10 minutes in PBS 0.1 M on a shaker.
11. Prepare ABC solution  at least 30 minutes prior to incubation to allow for ABC complex to form. Add 2 drops of solution A and 2 drops of solution B per 10 ml of blocking solution. Solutions A and B can also be added to plain PBS 0.1 M.
12. Incubate sections in ABC solution for 1-2 hours at room temperature on a shaker.
13. Rinse sections 4 x 10 minutes with PBS 0.1 M on a shaker.
14. Incubate sections in DAB solution for 8 minutes at room temperature on a shaker. DAB solution is highly toxic and carcinogen. Wear gloves and handle with care.
15. Add three drops of 0.3%  H₂O₂ (~125 ul) to each well to reveal staining. When background is satisfactory (after 1 to 5 min), halt the reaction by adding PBS 0.1 M.
16. Rinse sections 4 x 10 minutes with PBS 0.1 M on a shaker.
17. Transfer sections to slides using a brush, allow to air dry. It is best to transfer sections as soon as possible but well plates can be stored for a few days in the fridge at 4°C.
18. Dehydrate slides twice in ethanol 100% for 5 minutes each.
19. Incubate slides twice in toluene or xylene for 5 minutes each.
20. Add mounting medium to slides while still wet. Place coverslips to slides and allow to dry. Examine staining by microscopy.

Reagents

• Sodium phosphate, monobasic anhydrous NaH₂PO₄ (FW 120.0). Sigma,  S-0751, 1Kg
• Sodium phosphate, dibasic anhydrous, Na₂HPO₄ (FW 142.0). Sigma, S-0876, 1Kg
• Hydrogen peroxide, H2O2  30% (w/w) solution. Sigma, H-1009, 100 ml
• Albumin Bovine fraction V, min 96%, electrophoresis. Sigma, A-9647, 50g
• 3,3′-diaminobenzidine tablets (DAB). Sigma, D-5905, 50 tablets
• Goat serum. BioWest, Cat# S2000, 100ml or similar
• Vectastain ABC Kit, Elite standard. Vector, PK-6100
• Triton X-100 (t-Octylphenoxypolyethoxyethanol). Sigma, T-9284, 100 ml
• Toluene or xylene from VWR or Fisher
• Ethanol 100%

Antibodies

Titrate new batches of antibodies for appropriate concentration before using in experiments as effective concentrations may vary across batches of antibody?.

Primary

Rabbit anti-Fos polyclonal IgG, Oncogene Research Products (Ab-5, Cat.# PC38). Recommended dilution, 1:20 000.

Secondary

Biotin-SP-conjugated affiniPure Goat anti-rabbit IgG (H+L) (minimal cross reaction to Human , Mouse and rat serum proteins). Made in goat. Jackson Immunoresearch, Cat.# 111-065-144. Recommended dilution: 1:2000.

Solutions

Phosphate buffer solution, 0.2 M,  pH 7.4

1. Collect 1000 ml of distilled water in a graduated cylinder. Pour about 400 ml of water in a beaker and stir.
2. Weigh 4.8 g of Sodium Phosphate monobasic NaH₂PO₄ and 22.72 g of sodium phosphate dibasic Na₂HPO₄ .
3. Add to the 400 ml of water. When dissolved, add the rest of the water and continue stirring for 5 min. Take pH which should be around 7.4.

Phosphate buffer solution, 0.1 M,  pH 7.4

Make phosphate buffer 0.2M solution as described above and add 1000 ml of distilled water to bring it to 0.1 M, total volume 2 liters. pH should be around 7.4. Solution can be kept at room temperature or at 4°C.

Blocking solution (PBS 0.1 M; 0.1 % BSA; 0.2% Triton X-100; 2% serum)

Collect about 800 ml of phosphate buffer 0.1 M in a graduated cylinder. Add 20 ml of serum, 2 ml of Triton X-100 and 1 g of BSA. Stir for 10 min. Add more PBS 0.1 M to reach 1000 ml. Stir another 5 min. Store blocking solution in 50-ml aliquots (50-ml Falcon tubes) at -20°C

DAB solution, 0.05% (w/v)

Add 1 tablet (10 mg) of DAB in 20 ml of PBS 0.1 M in a 50-ml Falcon tube. Vortex vigorously until dissolved. Solution should be used fresh, or may be frozen in single-use aliquots and stored at -20C until use. Wear gloves and inactivate solution using a 10% bleach solution (dilute DAB with an equal volume of bleach) when finished and dispose in appropriate biohazard container. DAB is highly toxic and carcinogen; do not dump solution down the drain without treatment.

Neutralization of DAB

Although chlorine bleach is commonly employed in many laboratories as a neutralization procedure, it is not effective in removing the mutagenic properties of DAB. A potassium permanganate-sulfuric acid procedure must be used.

1. Take up bulk quantities of diaminobenzidine tetrahydrochloride dehydrate in water and bulk quantities of the free base in 0.1 M hydrochloric acid so that the concentration of DAB does not exceed 0.9 mg/ml.  Dilute solutions with the same buffer, if necessary, so that the concentration does not exceed 0.9 mg/ml.
2. For each 10 ml of solution, add 5 ml of 0.2 M potassium permanganate solution and 5 ml of 2 M sulfuric acid solution.
3. Allow the mixture to stand overnight, decolorize by the addition of sodium ascorbate, neutralize and dispose solution down the drain with copious amounts of water.

0.3% (v/v) H₂O₂ solution

Add 0.5 ml of H₂O₂ 30% solution to 50 ml of PBS 0.1 M in a 50-ml Falcon tube. Vortex. Use fresh.

Equipment

• Microscope
• 2D Shaker
• 6-well plates
• Gelatin-coated slides or precleaned superfrost plus slides (25 x 75 x 1 mm). VWR, Cat.# 48311-703
• Coverlips (micro cover glasses) 24 x 60 mm, No. 1. VWR, Cat.# 48404 454.
• Mounting medium (Eukit or Cytoseal 280 from Richard-Allan Scientific (8311-4) or similar)

Phosphate buffered saline (abbreviated as PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

Applications

PBS has many uses because it is isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse containers containing cells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface. The thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers may be used for the same purpose but with less effectiveness. PBS can be used to take a reference spectrum when measuring the protein adsorption in ellipsometry.

Additives can be used to add function. For example, PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. For these types of applications, Good’s buffers are recommended.

Preparation

There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium^[1]. One of the most common preparations is described below.

A 10 liter stock of 10x PBS can be prepared by dissolving 800 g NaCl, 20 g KCl, 144 g Na₂HPO₄ · 2H₂O and 24 g KH₂PO₄ in 8 L of distilled water, and topping up to 10 L. The pH is ~6.8, but when diluted to 1x PBS it should change to 7.4. When making buffer solutions, it is good practice to always measure the pH directly using a pH meter. If necessary, pH can be adjusted using hydrochloric acid or sodium hydroxide.

On dilution, the resultant 1x PBS should have a final concentration of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, and a pH of 7.4.
Another preparation is described in Molecular Cloning by Sambrook, Fritsch and Maniatis, Apendix B.12^[2] as follows:

For 1 litre of 1X PBS, prepare as follows:

1. Start with 800 ml of distilled water:
2. Add 8 g of NaCl.
3. Add 0.2 g of KCl.
4. Add 1.44 g of Na₂HPO₄.
5. Add 0.24 g of KH₂PO₄.
6. Adjust the pH to 7.4 with HCl.
7. Add distilled water to a total volume of 1 liter.

Dispense the solution into aliquots and sterilize them by autoclaving (20 min, 121°C, liquid cycle). Store at room temperature.

References

1. Dulbecco, R. et al. (1954): Plaque formation and isolation of pure lines with poliomyelitis viruses. In: J. Exp. Med. vol. 99 (2), pp. 167-182. PMID 13130792
2. Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, volume 3, apendix B.12
3. Portions of this article are from “Phosphate buffered saline. In Wikipedia, the free encyclopedia. Retrieved September 17, 2008, from http://en.wikipedia.org/wiki/Phosphate_buffered_saline.” This article has been reviewed for scientific accuracy and is used in accordance with Wikipedia’s GNU Free Documentation License (GFDL).