2009 Mayıs | Turkish Chemistry - Part 2
May 17

RIP (radio-immune precipitation)

RIP (radio-immune precipitation)

Radio-immune precipitation (RIP) can take over where a Western blot will fail you. Western blots let you know how much protein has accumulated in a sample. If you are more interested in the rate of synthesis of protein in a , or if your protein degrades too quickly to be detected by a Western blot, then RIP is definitely a technique you’ll want to know about. RIP also detects protein-protein interaction, while Western blotting can’t.

This chart should help illustrate the differences between RIP and Western blotting.

 

Let’s look at this technique in greater detail.

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    • [35S] labels methionine—high energy, high specificity
    • [3H] labels amino acids—low energy
    • [14C] labels amino acids—low specific activity 

    • 1. Begin by radioactively labeling your cells.

    You have the following choices:

     

    2. Extract-release your protein mixture. You’ll need to make antibodies against one protein, then incubate your protein in them. This will give you an antibody-protein complex, but it will also leave you with some free antibodies floating around. So you’ll need to purify your antibody-protein complexes.

    3. Use the bacteria staphlococcus aureus in excess to purify your protein. Before incubating your protein in staph aureus, fix it with formaldehyde or glutaraldehyde to kill the staph aureus, since it’s a pathogen. The formaldehyde or glutaraldehyde will crosslink all of the proteins in the staph aureus . Staph aureus has on its surface a protein that binds to the tail of antibodies (to the Fc portion of the Ig ). That means it binds both free antibodies and your radiolabeled antibody-protein complexes.

    4. Centrifuge the solution to get a precipitate. Discard the solution and save the pellet. Elute the antibody-protein complexes by boiling them in SDS sample buffer. This denatures the proteins and removes the antibodies from the staph aureus and the radiolabled proteins.

     5. Run this on an SDS gel. Perform an autoradiograph and develop the x-ray film. You’ll see a dark spot on the film for whatever protein was bound by the antibody.

     

May 17

PCR (polymerase chain reaction)

Let’s say you have a biological sample with trace amounts of DNA in it. You want to work with the DNA, perhaps characterize it by sequencing, but there isn’t much to work with. This is where PCR comes in. PCR is the amplification of a small amount of DNA into a larger amount. It is quick, easy, and automated. Larger amounts of DNA mean more accurate and reliable results for your later techniques.

The techniques was developed by Nobel laureate biochemist Kary Mullis in 1984 and is based on the discovery of the biological activity at high temperatures of DNA polymerases found in thermophiles (bacteria that live in hot springs).Most DNA polymerases (enzymes that make new DNA) work only at low temperatures. But at low temperatures, DNA is tightly coiled, so the polymerases don’t stand much of a chance of getting at most parts of the .

But these thermophile DNA polymerases work at 100C, a temperature at which DNA is denatured (in linear form). This thermophilic DNA polymerase is called Taq polymerase, named after Thermus aquaticus, the bacteria it is derived from.

Taq polymerase, however, has no proofreading ability. Other thermally stable polymerases, such as Vent and Pfu, have been discovered to both work for PCR and to proofread.

You’ll need four things to perform PCR on a sample:

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      1. The target sample. This is the biological sample you want to amplify DNA from.

      2. 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.

      3. Taq polymerase. This is the enzyme that is in charge of replicating DNA. This is the polymerase part of the name polymerase chain reaction.

      4. Nucleotides. You’ll need to add nucleotides (dNTPs) so the DNA polymerase has building blocks to work with.

There are three major steps to PCR and they are repeated over and over again, usually 25 to 75 times. This is where the automation is most appreciated.

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          1. Annealing temperature. Starts at the low end of what you think will work, then move up as necessary. If the temperature is too low, the primers will make more mistakes and you’ll get too many bands when you run your sample on a gel. If the temperature is too high you will get no results and your gel will be blank. You want to be about 3C to 5C below the melting temperature (Tm). A rough formula for determining Tm is Tm=4(G + ) + 2(A + T).

          2. Magnesium concentration. You want your Mg2+ concentration to be about 1.5mM to 3mM. If you go too high, the polymerase will make more mistakes.

          3. Think carefully about primer design. Both primers should have approximately the same Tm so they both anneal at the same temperature. Two out of three bases on the 3′ end should b G or to get good hybridization (G and have three H-bonds so you get better polymerization). Lastly, avoid primer dimers, which occur when the primers have ends that will anneal to each other. This will produce NO product.

          4. More is not necessarily better. More polymerase produces more nonspecific product, so don’t just carelessly dump in a bunch of polymerase. Additionally, PCR reactions don’t work if there is too much DNA.

      • 1. Your target sample is heated. This denatures the DNA, unwinding it and breaking the bonds that hold together the two strands of the DNA molecule, leaving you with single stranded DNA (ssDNA).

      2. Temperature is reduced and the primer is added. The primer 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.

      3. 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. The job of Taq polymerase is to move along the strand of DNA and use it as a template for assembling a new stand that is complimentary to the template. This is the chain reaction in the name polymerase chain reaction.

      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.

      Perhaps the most important thing to remember is to be very aware of contamination. If, for example, you unknowingly slough off a piece of skin into your sample, then your DNA may be amplified in the PCR reaction.  Here are some other factors to optimize your results with PCR:

      RT-PCR

      Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA . The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA . After you add your RNA sample to the PCR machine, add a DNA primer as usual and allow it to anneal to your target molecule. Then add RT along with dNTPs, which will elongate the DNA primer and make a cDNA copy of the RNA and 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.

May 14

Southern blotting

Southern blotting

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an onto a membrane. Southern blotting is designed to locate 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. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

dSouthern blot

Let’s look at this technique in greater detail.  

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    • 1. Digest the DNA with an appropriate restriction enzyme.

    2. Run the digest on an .

    3. Denature the DNA (usually while it is still on the gel).
    For example, soak it in about 0.5M NaOH, which would separate  double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.

    A depurination step is optional. Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. Be aware, however, that the procedure may also be hampered by fragments that are too small.

    Be sure to neutralize the after this step, or the base after the prior step if you don’t depurinate.

    Transfer DNA to membrane4. Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is less fragile. Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.

    You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, excepts more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)

    After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)

    5. Probe the membrane with labeled ssDNA. This is also known as hybridization.
    Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands.
    Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.

    A prehybridization step is required before hybridization to block non-specific sites, since you don’t want your single-stranded probe binding just anywhere on the membrane.

    To hybridize, use the same buffer as for prehybridization, but add your specific probe.

    6. Visualize your radioactively labeled target sequence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. Biotin/streptavidin detection is done by colorimetric methods, and bioluminescent visualization uses luminesence.

32P labeled ATP
Treat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which causes double-stranded nicks in DNA. Add 32P, dATP, and other dNTPs to DNA polymerase I, which has 5′ to 3′ polymerase activity and 5′ to 3′ exonuclease activity.

Nick translation occurs and as the nick is translated down the DNA strand, the polymerase activity continues to nick while the exonuclease activity continues to fill in the nick. As this happens, 32P becomes incorporated into, and thus labels, the DNA. Heat the DNA to make it single stranded, then immediately place it on ice to keep the two strands from reannealing to each other. (If the DNA is on ice, the DNA passes through the annealing temperature too quickly for the DNA to rehybridize into double-stranded DNA.)

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