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15.6: Exercise 2 - Immunodetection - Biology

15.6: Exercise 2 - Immunodetection - Biology


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This is a multi-day procedure. Membranes are rehydrated and treated with blocking reagents

  1. Wearing GLOVES, unwrap the dry blot from the plastic wrap. Use the prestained standards to identify the side of the membrane to which the proteins are bound. Submerge the membrane in methanol with this side facing up. Gently agitate the membrane by hand rocking for 30-60 seconds until the membrane has been uniformly wet with methanol. Decant the methanol into the appropriate container and fill the tray half way full with deionized water. Gently agitate the membrane for an additional minute.
  2. Decant the water and replace it with sufficient TBS-T (Tris buffered-saline containing 0.05% Tween 20) to cover the blot. Place the blot on a rocking platform. Equilibrate the blot in TBS-T for 5 minutes with slow rocking. At the end of 5 minutes, drain off the TBS-T.
  3. Pour enough blocking solution (5% nonfat milk in TBS-T) onto the blot to cover it.
  4. Cover the tray with a small piece of plastic wrap. Label the tray clearly and place the tray on a rocking platform in the cold room. The blot should float freely in the tray so that both sides are washed. Incubate the blot for at least an hour or up to 24 hours at 4 ̊C.

Membranes are washed and incubated with primary antibody (~24 hours)

  1. Locate your blot in the cold room and bring it back to the lab room.
  2. Remove the plastic wrap from the container holding the blot and pour off the blocking solution. SAVE the plastic wrap! You will need it to cover the container again!
  3. Add enough TBS-T to cover the blot and place the container on the rocking platform. Rock for 5 minutes.
  4. Pour off the TBS-T. Add 15 mL of primary antibody diluted in blocking buffer.
  5. Cover the container with the same piece of plastic wrap and place the tray on the rocking platform in the 4 ̊C cold room. Make sure that the blot floats freely in the tray and that the standards are on the top face of the blot. Incubate overnight at 4 ̊C with slow rocking. NOTE: The timing of this step is the most critical in the procedure. Shortening the incubation time with primary antibody may reduce the sensitivity of the western blot.

Secondary antibody binding and detection (1.5-2 hours)

  1. Locate your blot in the cold room and bring it to your lab classroom.
  2. Carefully drain the antibody from the blot into the test tube marked “Used primary antibody”. (Antibodies are expensive. Fortunately, the solutions can be re-used.)
  3. Fill the tray with the blot about half-full with TBS-T. Place the tray on a rocking platform and wash the membrane for 5 minutes to remove unbound primary antibody. Drain the TBS-T when the wash is complete.
  4. Repeat step 3 once more, for a total of two washes.
  5. Add enough secondary antibody to cover the blot and incubate the membrane for 1 hour with gentle rocking at room temperature. The secondary antibody, which is conjuated to horseradish peroxidase (HRP), has been diluted in TBS-T.
  6. Carefully drain the antibody from the blot into the test tube marked “Used secondary antibody.”
  7. Wash the membrane 3 times for 5 minutes each with TBS-T, as in step 3.
  8. Drain the TBS-T from the blot. Using a P1000 micropipette, cover the blot with 1 mL of 3,3’5,5’-tetramethyl benzidine (TMB), a colorigenic substrate for HRP. Let the color continue to develop until distinct bands are apparent. Bands will probably become apparent within minutes. Do not allow the blot to over-develop, when nonspecific bands become apparent.

9. Stop color development by diluting the substrate with an excess of deionized water. Drain the diluted substrate into the waste container.

10. Allow the blot to dry on a piece of filter paper. Record your data with your cell phone camera.


Diet and Exercise: a Match Made in Bone

Purpose of review: Multiple dietary components have the potential to positively affect bone mineral density in early life and reduce loss of bone mass with aging. In addition, regular weight-bearing physical activity has a strong positive effect on bone through activation of osteocyte signaling. We will explore possible synergistic effects of dietary components and mechanical stimuli for bone health by identifying dietary components that have the potential to alter the response of osteocytes to mechanical loading.

Recent findings: Several (sub)cellular aspects of osteocytes determine their signaling towards osteoblasts and osteoclasts in response to mechanical stimuli, such as the osteocyte cytoskeleton, estrogen receptor α, the vitamin D receptor, and the architecture of the lacunocanalicular system. Potential modulators of these features include 1,25-dihydroxy vitamin D3, several forms of vitamin K, and the phytoestrogen genistein. Multiple dietary components potentially affect osteocyte function and therefore may have a synergistic effect on bone health when combined with a regime of physical activity.

Keywords: Bone health Diet Dietary components Nutrition Osteocytes Physical activity.

Conflict of interest statement

Conflict of Interest

Ellen GHM van den Heuvel and Ruud JW Schoemaker are employees of FrieslandCampina, a dairy company.

Astrid Bakker, Jenneke Klein-Nulend, and Hubertine Willems declare no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.


Scientists discover a natural molecule to treat type 2 diabetes: Molecule mimics some effect of physical exercise

Researchers at the Université Laval Faculty of Medicine, the Quebec Heart and Lung Institute Research Center, and the Institute of Nutrition and Functional Foods have discovered a natural molecule that could be used to treat insulin resistance and type 2 diabetes. The molecule, a derivative of omega-3 fatty acids, mimics some of the effects of physical exercise on blood glucose regulation.

The details of the discovery made by Professor André Marette and his team are published today in Nature Medicine.

It has been known for some time that omega-3 fatty acids can help reduce insulin resistance caused by a diet high in saturated fat. In their earlier work, André Marette and his colleagues had linked these effects to a bioactive lipid called protectin D1. In investigating further, they discovered that another member of the same family named protectin DX (PDX) triggers the production and release of interleukin 6 (IL-6) in muscle cells, a response that also occurs during physical exercise. "Once in the bloodstream, IL-6 controls glucose levels in two ways: it signals to the liver to reduce glucose production and acts directly on the muscles to increase glucose uptake," explains the researcher who is also Scientific Director of Université Laval's Institute of Nutrition and Functional Foods.

The researchers used transgenic mice lacking the IL-6 gene to demonstrate the link between PDX and IL-6. PDX had very little effect on the control of blood glucose in these animals. In similar tests conducted on obese diabetic rats, PDX was shown to dramatically improve responsiveness to insulin, the hormone which regulates blood glucose. "The mechanism of action described for PDX represents a new therapeutic strategy for improving glucose control," proposes the researcher. "Its efficacy may be comparable with that of certain drugs currently prescribed to control glycemia."

Even though PDX appears to mimic the effect of physical exercise by triggering IL-6 secretion in the muscles, André Marette warns that it is not a substitute for physical activity. "Exercise has cardiovascular and other hormonal benefits that go well beyond its metabolic effects on the muscles," adds the researcher whose work is supported by the Canadian Institutes of Health Research (CIHR) and the Canadian Diabetes Association.

Professor Marette and Université Laval have filed a patent application for PDX and its therapeutic applications. "For us, the next step is to demonstrate the antidiabetic effects in humans and determine the receptor through which PDX acts."

In addition to André Marette, the study is authored by Phillip White, Philippe St-Pierre, Alexandre Charbonneau, Patricia Mitchell, Emmanuelle St-Amand, and Bruno Marcotte.


Diet and Exercise: a Match Made in Bone

Purpose of review: Multiple dietary components have the potential to positively affect bone mineral density in early life and reduce loss of bone mass with aging. In addition, regular weight-bearing physical activity has a strong positive effect on bone through activation of osteocyte signaling. We will explore possible synergistic effects of dietary components and mechanical stimuli for bone health by identifying dietary components that have the potential to alter the response of osteocytes to mechanical loading.

Recent findings: Several (sub)cellular aspects of osteocytes determine their signaling towards osteoblasts and osteoclasts in response to mechanical stimuli, such as the osteocyte cytoskeleton, estrogen receptor α, the vitamin D receptor, and the architecture of the lacunocanalicular system. Potential modulators of these features include 1,25-dihydroxy vitamin D3, several forms of vitamin K, and the phytoestrogen genistein. Multiple dietary components potentially affect osteocyte function and therefore may have a synergistic effect on bone health when combined with a regime of physical activity.

Keywords: Bone health Diet Dietary components Nutrition Osteocytes Physical activity.

Conflict of interest statement

Conflict of Interest

Ellen GHM van den Heuvel and Ruud JW Schoemaker are employees of FrieslandCampina, a dairy company.

Astrid Bakker, Jenneke Klein-Nulend, and Hubertine Willems declare no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.


Exercise inhibits tumor growth and central carbon metabolism in patient-derived xenograft models of colorectal cancer

Background: While self-reported exercise is associated with a reduction in the risk of recurrence in colorectal cancer, the molecular mechanisms underpinning this relationship are unknown. Furthermore, the effect of exercise on intratumoral metabolic processes has not been investigated in detail in human cancers. In our current study, we generated six colorectal patient patient-derived xenografts (CRC PDXs) models and treated each PDX to voluntary wheel running (exercise) for 6-8 weeks or no exposure to the wheel (control). A comprehensive metabolomics analysis was then performed on the PDXs to identify exercise induced changes in the tumor that were associated with slower growth.

Results: Tumor growth inhibition was observed in the voluntary wheel running group compared to the control group in three of the six models. A metabolomics analysis first revealed that central carbon metabolism was affected in each model irrespective of treatment. Interestingly, comparison of responsive and resistant models showed that levels of metabolites in nucleotide metabolism, known to be coupled to mitochondrial metabolism, were predictive of response. Furthermore, phosphocreatine levels which are linked to mitochondrial energy demands were associated with inhibition of tumor growth.

Conclusion: Altogether, this study provides evidence that changes to tumor cell mitochondrial metabolism may underlie in part the benefits of exercise.

Keywords: Central carbon metabolism Colorectal cancer Exercise Mitochondrial metabolism Patient-derived xenograft.


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