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Molecular analysis of proteins

In many cases it may not be desirable or possible to study DNA or RNA directly. Proteins can provide species-specific information for identification as well as important information about how and whether a cell or tissue is responding to the presence of a pathogenic microorganism. Various proteins require different methods for isolation and characterization.

Polyacrylamide gel electrophoresis

A variation of gel electrophoresis, called polyacrylamide gel electrophoresis (PAGE) , is commonly used for separating proteins. In PAGE , the gel matrix is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is typically performed using a vertical gel apparatus ( [link] ). Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS) , masking the native charges and allowing separation based on size only. PAGE can be further modified to separate proteins based on two characteristics, such as their charges at various pHs as well as their size, through the use of two-dimensional PAGE . In any of these cases, following electrophoresis, proteins are visualized through staining, commonly with either Coomassie blue or a silver stain.

a) A diagram showing a globular protein with positive and negative charges undergoing SDS treatment. SDS denatures the protein (producing a linear product) and makes them uniformly negative in charge. B) The protein samples are then placed into the wells of an SDS_PAGE gel. One well is loaded with a molecular weight standard. The gel is then exposed to a power source that results in the top of the gel (near the wells) becoming negative charged and the bottom becoming positively charged. Proteins migrate through the gel from the negative to the positive sides. Small proteins travel through the gel faster than large proteins. The molecular weight standard includes fragments of known size and is used to estimate the size of sample proteins. In this example the standard has sizes of 216, 132, 78, 32 and 7. The other lanes have bands of various sizes. C) A photograph of an SDS-PAGE gel. Purple bands on a clear background.
(a) SDS is a detergent that denatures proteins and masks their native charges, making them uniformly negatively charged. (b) The process of SDS-PAGE is illustrated in these steps. (c) A photograph of an SDS-PAGE gel shows Coomassie stained bands where proteins of different size have migrated along the gel in response to the applied voltage. A size standard lane is visible on the right side of the gel. (credit b: modification of work by “GeneEd”/YouTube)
  • On what basis are proteins separated in SDS-PAGE?

Part 3

When Kayla described her symptoms, her physician at first suspected bacterial meningitis , which is consistent with her headaches and stiff neck. However, she soon ruled this out as a possibility because meningitis typically progresses more quickly than what Kayla was experiencing. Many of her symptoms still paralleled those of amyotrophic lateral sclerosis (ALS) and systemic lupus erythematosus (SLE) , and the physician also considered Lyme disease a possibility given how much time Kayla spends in the woods. Kayla did not recall any recent tick bites (the typical means by which Lyme disease is transmitted) and she did not have the typical bull’s-eye rash associated with Lyme disease ( [link] ). However, 20–30% of patients with Lyme disease never develop this rash, so the physician did not want to rule it out.

Kayla’s doctor ordered an MRI of her brain, a complete blood count to test for anemia, blood tests assessing liver and kidney function, and additional tests to confirm or rule out SLE or Lyme disease. Her test results were inconsistent with both SLE and ALS, and the result of the test looking for Lyme disease antibodies was “equivocal,” meaning inconclusive. Having ruled out ALS and SLE, Kayla’s doctor decided to run additional tests for Lyme disease.

  • Why would Kayla’s doctor still suspect Lyme disease even if the test results did not detect Lyme antibodies in the blood?
  • What type of molecular test might be used for the detection of blood antibodies to Lyme disease?
A photo of a bulls-eye rash; a red spot in the center and a red ring around that.
A bulls-eye rash is one of the common symptoms of Lyme diseases, but up to 30% of infected individuals never develop a rash. (credit: Centers for Disease Control and Prevention)

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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