1

Cellular and Cellular and Molecular Molecular Biology Biology

TechniquesTechniques

2

Cell CultureCell Culture• Tissue Culture is the general term for the

removal of cells, tissues, or organs from an animal

• their subsequent placement into an artificial environment - a suitable culture vessel containing a liquid or semisolid medium - nutrients essential for survival and growth.

3

WORK AREA AND WORK AREA AND EQUIPMENT EQUIPMENT

• Laminar flow hoods - continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air

• CO2 Incubators. an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained. Culture flasks should have loosened caps for gas exchange

• Microscopes. Inverted phase contrast microscopes • Vessels. Anchorage dependent cells require a

nontoxic, biologically inert, and optically transparent surface that will allow cells to attach and mouve - polystyrene plastic - supplied sterile and disposable

4

Primary CulturePrimary Culture• When cells are surgically removed from an

organism and placed into a suitable culture environment, they will attach, divide and grow.

• There are two basic methods. – Explant Cultures, small pieces of tissue are

attached to a culture vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant out onto the culture vessel surface

– Enzymatic Dissociation, uses digesting (proteolytic) enzymes, such as trypsin or collagenase, to dissolve the cement holding the cells together - a suspension of single cells that are then placed into culture vessels containing culture medium

5

Cell culture phasesCell culture phases• Lag Phase - usually the first 1-2 days, there is little or

no increase in cell number. The cells undergo internal cytoskeletal and enzyme changes and adjust to the new media.

• Log Phase - the cell number increases exponentially. This growth will continue as long as there are sufficient nutrients to support the increasing cell number. Eventually some critical nutrient will become limiting, however.

• Plateau Phase - the number of cells remains constant. Eventually, the cells will die unless subcultured or fresh media is added. The cells will continue to grow in contact with the vessel and give rise to a "monolayer" culture. The cells will cease to divide when they reach confluency; they demonstrate contact inhibition.

6

SubculturingSubculturing• Ideally, cells are harvested when they are in a semi-

confluent state and are still in log phase • This is usually done by removing them as gently as

possible from the substrate with enzymes. • Some cell lines can be harvested by gently scraping

the cells off from the bottom of the culture vessel. • Once released, the cell suspension can then be

subdivided and placed into new culture vessels.• Once a surplus of cells is available, they can be

treated with suitable cryoprotective agents, such as dimethylsulfoxide (DMSO), carefully frozen and then stored at cryogenic temperatures (below -130°C)

7

Media and growth requirementsMedia and growth requirements • Physiological parameters

– pH - 7.2-7.5 and osmolality of medium must be maintained– temperature - 37ºC– humidity is required– gas phase - bicarbonate conc. and CO2 tension in equilibrium – Away from visible light - light induces production of toxic compounds in

some media; • Medium requirements: (often empirical)

– Ions - Na, K, Ca, Mg, Cl, P, Bicarbonate – Trace elements - iron, zinc, selenium– sugars - glucose is the most common– amino acids - essential– vitamins - B, etc.– choline, inositol– serum - contains a large number of growth promoting activities such as

buffering toxic nutrients by binding them, neutralizes trypsin and other proteases, has undefined effects on the interaction between cells and substrate, and contains peptide hormones or hormone-like growth factors that promote healthy growth.

– antibiotics - not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.

• Feeding - 2-3 times/week.

8

9

Cell Fractionation Cell Fractionation • chemical knowledge of organelle function by

isolating organelles into reasonably pure fractions

• Each organelle has characteristics (size, shape and density for example) which make it different from other organelles within the same cell

• The process of breaking open cells is homogenization

• the subsequent isolation of organelles is fractionation

10

HomogenizationHomogenization • Cells which are part of a more solid tissue (such

as liver or kidney) will first need to be separated from all connections with other cells

• need to be enzymatically or mechanically disaggregated

• Homogenization techniques can be divided into– those brought about by osmotic alteration of the

media which cells are found in– those which require physical force to disrupt cell

structure : use of blenders, or ultrasonification

11

Osmotic alterationsOsmotic alterations • Many organelles are easier to separate if the

cells are slightly swollen. • The inbibition of water into a cell will cause

osmotic swelling of the cell - rupture of the cell membrane and subsequent organelle separation.

• The use of a hypo-osmotic buffer can be very beneficial, for example, in the isolation of mitochondria and in the isolation of mitotic chromosomes.

12

UltrasonicationUltrasonication • Ultrasonicators have been used to

separate organelles from cells, particularly from tissue culture cells.

• Light use of an ultrasonic wave can readily remove cells from a tissue culture substrate (such as the culture flask).

• It can also be adjusted to separate cells, or to break open the plasma membrane and leave the internal organelles intact

13

Cell fractionation allows the isolation of cell constituents by differential centrifugation. The drawings at right show the cellular organelles at the bottom of each tube after centrifugation. Centrifugal force is expressed by g, which is equivalent to the force of gravity. (1) A fragment of tissue is minced and dissociated with a homogenizer or by ultrasound. (2) The dissociated tissue is left standing for about 20 min. Clumps of cells and fibers of extracellular matrix precipitate to the bottom. (3) The supernatant is centrifuged at 1000 g for 20 min. Nuclei precipitate. (4) The supernatant is centrifuged at 10,000 g for 20 min. Mitochondria and lysosomes precipitate. (5) The supernatant is centrifuged at 105,000 g for 120 min. Microsomes precipitate. (6) If the supernatant is first treated with sodium deoxycholate and then centrifuged at 105,000 g for 120 min, the microsomes dissociate and precipitate separately as endoplasmic reticulum membranes and ribosomes.

14

Electron micrographs of 3 cell fractions isolated by density gradient centrifugation. A: Mitochondrial fraction, contaminated with microsomes. B: Microsomal fraction. C: Lysosomal fraction. High magnifications. (Courtesy of P Baudhuin.)

15

PROTEIN PROTEIN ISOLATIONISOLATION

16

Ultracentrifugation (A) and chromatography (B): methods of protein isolation. A: A mixture of proteins obtained from homogenized cells or tissues is submitted to centrifugation at high speed for several hours. The proteins separate into several bands, depending on the size and density of the protein molecules. The ultracentrifugation medium is drained and collected in several fractions that contain different proteins, which can be analyzed further. B: A mixture of proteins obtained from homogenized cells or tissues is added to a column filled with particles that have different chemical properties - different electrostatic charges (attracting proteins according to their charge) or different sizes of pores (acting as sieves for different-sized molecules). As the proteins migrate through the column, their movement is slowed according to their interaction with the particles. When the effluent is recovered, the different groups of proteins may be collected separately.

17

Ion exchange chromatographyIon exchange chromatography • based on the charge of the protein you are trying to

isolate.

• If the protein has a high positive charge, you'll pass it through a column with a negative charge or, you can bind a negatively charged protein to a positively charge column

• The charge on the column will bind the charged protein, and other proteins will pass through.

• to release your positively charged protein from the negatively charged column - a cation exchange column uses sulfonated residues.

• positively charge column is called an anion exchange column - uses quaternary ammonium residues.

18

Gel filtration chromatography Gel filtration chromatography • separates proteins on the basis of their size.

The column is packed with a matrix of fine porous beads

• The beads have very small holes.

• As the protein solution is poured on the column, small molecules enter the pores in the beads.

• Larger molecules are excluded from the holes, and pass quickly between the beads

19

20

Gel electrophoresis: a method of protein isolation. A: Isolation of proteins. (1) Mixtures of proteins are obtained from homogenized cells or tissues. They are usually treated with a strong detergent (sodium dodecyl sulfate) and with mercaptoethanol to unfold and separate the protein subunits. (2) The samples are put on top of a polyacrylamide gel, which is submitted to an electrical field. The proteins migrate along the gel according to their size and shape. (3) A mixture of proteins of known molecular mass is added to the gel as a reference to identify the molecular mass of the other proteins. B: Detection and identification of the proteins.(1) Staining. All proteins will stain the same color. The color intensity is proportional to the protein concentration. (2) Autoradiography. Radioactive proteins can be detected by autoradiography. An x-ray film is apposed to the gel for a certain time and then developed. Radioactive proteins will appear as dark bands in the film. (3) Immunoblotting. The proteins can be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with an antibody made against proteins that may be present in the sample.

21

SDS-PAGE SDS-PAGE • SDS-PAGE stands for sodium dodecyl sulfate-

polyacrylamide gel electrophoresis

• SDS portion it is an anionic detergent that binds quantitatively to proteins, giving them linearity and uniform charge, so that they can be separated solely on the basis of their size

• The number of SDS molecules that bind to a protein is proportional to the number of amino acids that make up the protein.

• Each SDS molecule contributes two negative charges, overwhelming any charge the protein may have.

• SDS also disrupts the forces that contribute to protein folding, ensuring that the protein is not only uniformly negatively charged, but linear as well

22

• polyacrylamide gel electrophoresis separates protein molecules according to their size.

• is a cross-linked matrix that functions as a sort of sieve to help "catch" the molecules

• an electric current is used to move the protein molecules across a polyacrylamide gel.

• The smaller molecules are able to navigate faster than the larger one, so they make it further down the gel

• Once an SDS-PAGE gel is run, fix the proteins in the gel so they don't come out when you stain the gel. Acetic acid 25% in water is a good fixative, as it keeps the proteins denatured.

• The gel is typically stained with Coomasie blue dye R250

23

Western Blot Western Blot • Western blot analysis can detect one

protein in a mixture of proteins while giving you information about the size of the protein.

• Western blotting tells you how much protein has accumulated in cells

24

• Separate the proteins by size using SDS-PAGE • Place a nitrocellulose membrane on the gel

and, using electrophoresis, drive the protein bands onto the nitrocellulose membrane drive the negatively charged proteins over to the positively charged nitrocellulose membrane

• Incubate the nitrocellulose membrane with a primary antibody

• Incubate with a secondary antibody. This antibody should be an antibody-enzyme conjugate - alkaline phosphatase

• To see the enzyme in action, incubate it with a substrate which will precipitate

• Put x-ray film on your gel to detect a flash of light, which is given off by the enzyme

25

26

RIP (radio-immune RIP (radio-immune precipitation)precipitation)

• If you are more interested in the rate of synthesis of protein in a cell, or if your protein degrades too quickly to be detected by a Western blot

• also detects protein-protein interaction, while Western blotting can't.

• Uses radioactivity

27

ELISA (Enzyme-Linked ELISA (Enzyme-Linked Immunosorbent Assay) Immunosorbent Assay)

• powerful method in estimating ng/ml to pg/ml of peptides, proteins, antibodies and hormones in the solution, such as serum, urine and culture supernatant.

• an antigen must be immobilized to a solid surface.

• The antigen is then recognized by an antibody that is linked to an enzyme.

• Detection is accomplished by incubating the enzyme-complex with a substrate that produces a detectable product.

28

• Most commonly, ELISAs are performed in 96-well (or 384-well) polystyrene plates

• The antigen is added to the wells where some remain adsorbed by hydrophobic association to the walls after washing away the excess

• During an infection, an individual mounts an antibody response - production of plasma IgG molecules that bind to various parts of the infectious agent.

• If these antibodies are present in the sample, they will bind to the adsorbed antigens in the well and remain there after washing

29

30

31

NUCLEIC ACIDSNUCLEIC ACIDSANALYZE ANALYZE

32

Southern blotting Southern blotting • named after Edward M. Southern who

developed this procedure at Edinburgh University in the 1970s

• DNA molecules are transferred from an agarose gel onto a membrane.

• locates a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome.

• The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific

33

34

• Digest the DNA with an appropriate restriction enzyme.

• Run the digest on an agarose gel • Denature the DNA separate  double-stranded DNA

into single-stranded DNA • Transfer the denatured DNA to the

membrane. a nitrocellulose membrane. Transfer is usually done by capillary action

• treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane.

• Probe the membrane with labeled ssDNA. Hybridization - ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands.

• Visualize the radioactively labeled target sequence by autoradiograph

35

Northern blotting Northern blotting • locating a sequence of RNA

• Northern hybridization or RNA hybridization

• The procedure is almost identical to that of Southern blotting, except you are working with RNA instead of DNA

36

PCR (polymerase chain PCR (polymerase chain reaction) reaction)

• a biological sample with trace amounts of DNA in it. You want to work with the DNA, characterize it by sequencing

• PCR is the amplification of a small amount of DNA into a larger amount.

• It is quick, easy, and automated • developed by Nobel laureate biochemist Kary

Mullis in 1984 and • based on the discovery of the biological activity

at high temperatures of DNA polymerases found in thermophiles

37

• Most DNA polymerases (enzymes that make new DNA) work only at low temperatures. But at low temperatures, DNA is tightly coiled

• thermophile DNA polymerases, called Taq polymerase, work at 100C, a temperature at which DNA is denatured (in linear form - named after Thermus aquaticus

• PCR is so efficient because it multiplies the DNA exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of DNA that is made can serve as a template for new ones

38

4 things to perform PCR 4 things to perform PCR

• The target sample. the DNA sample. • A primer. Short strands of DNA that adhere to

the target segment. They identify the portion of DNA to be multiplied and provide a starting place for replication.

• Taq polymerase. This is the enzyme that is in charge of replicating DNA. This is the polymerase part

• Nucleotides. (dNTPs) so the DNA polymerase has building blocks to work with.

39

3 major steps repeated 25 to 75 3 major steps repeated 25 to 75 timestimes

• Target sample is heated. This denatures the DNA, unwinding it and breaking the bonds that hold together the two strands - single stranded DNA (ssDNA).

• Temperature is reduced and the primer is added. The primer molecules now have the opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of DNA to be amplified and provides a starting place for replication.

• New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new pieces of ssDNA that are complimentary to the portions marked by the primers. This is the chain reaction in the name polymerase chain reaction.

40

41

• Then run an Agarose gel electroporesis to visualize the fragments

• separates different DNA molecules according to their size.

• The phosphate molecules that make up the backbone of DNA molecules have a high negative charge.

• When DNA is placed on a field with an electric current, these negatively charged DNA molecules migrate toward the positive end of the field

• The smaller molecules are able to navigate the mesh faster

• The gel is stained with ethidium bromide so you can visualize how these DNA molecules resolved into bands along the gel

42

RT-PCR RT-PCR • The incorporation of the enzyme reverse transcriptase

(RT), can be combined with traditional PCR to allow for the amplification of RNA molecules.

• add your RNA sample to the PCR machine,

• add a DNA primer as usual and allow it to anneal to your target molecule.

• add RT along with dNTPs, which will elongate the DNA primer and make a cDNA copy of the RNA molecules

• run the PRC reaction as usual.

• The product of RT-PCR is a double stranded DNA molecule analogous to the target segment of the RNA molecule.