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Autophagy is a lysosomal-dependent degradation pathway widely existing in eukaryotic cells. Its activation involves the formation of autophagosomes, the transport of autophagic substrates and their degradation in autophagic lysosomes. Creative Bioarray provides several dynamic detection services to study autophagy.

Background

In recent years, more and more researchers have realized that simply observing the number of autophagic lysosomes is not enough to judge the changes of autophagic flux, and detecting the degradation of long-lived proteins can be a good evaluation of the changes of autophagic flux.

Long-lived-Protein-Detection

Intracellular proteins are mainly degraded through two pathways, the proteasome pathway and the autophagy pathway. The proteasome pathway is mainly responsible for the degradation of short-lived proteins, while the long-lived proteins and some organelles are mainly degraded through the autophagy pathway. Under pathological conditions, the obstruction or over-activation of autophagy will cause abnormal degradation of long-lived proteins, which will result in the failure to maintain the normal function and morphology of cells.


Our Services

Dynamic detection can not only prove the existence of autophagy, but also reflect the entire dynamic process by detecting the degradation rate of autophagic proteins or the accumulation of autophagosomes. Our services include but are not limited to:

Long-lived Protein Detection Service

Observation of autophagy protein degradation is a previously established method for dynamic quantitative analysis of autophagy. Generally, radioactive amino acids are incorporated into cellular proteins, and protein degradation is quantified by detecting radiolabeled amino acids.

Experiment Process

  • The isotope-labeled amino acids are incubated with the cultured cells for a long time.
  • Wash the cells to remove radioactive materials that have not entered the cells.
  • Use non-isotopically labeled amino acids to incubate with cells for a short time
  • After the proteasome rapidly degrades the short-lived protein, replace the hungry medium HBSS or EBSS to induce autophagy.
  • Activate the culture for several hours, during which 3-methyladenine (3-MA) can be added to inhibit the formation of autophagic vesicles, thereby inhibiting autophagy.
  • Finally, trichloroacetic acid is added to the cells.
  • Detect the isotopes in the culture supernatant and the isotopes in the cells.
  • Calculating the ratio of the isotope in the supernatant to the isotope in the pelleted cells can give the degradation rate of long-lived protein.

Advantages

  • High sensitivity
  • Accurate and reliable
  • Wide application

Customer Notice

This method has low specificity and cannot distinguish between autophagy-dependent degradation and independent degradation. Therefore, it is usually necessary to add lysosomal antagonists, such as chloroquine, ammonium chloride and Baf A1. The specificity of this method was investigated by analyzing the release of isotope-labeled amino acids before and after adding the antagonist. Although this method has shortcomings, it can still accurately measure the amount of residual radioactive protein. Combined with other autophagy detection methods provided by Creative Bioarray, your research results can be more accurate and reliable.

Customers provide

  • Samples
  • Test purpose
  • Test requirements

We deliver

  • Reagents and instruments
  • Experiment process
  • Relevant raw and analysis data
  • Complete experiment report

Experiment cycle

The time depends on the experiment content

Advantages of Our Services

  • Complete instrument platform

    We have many types of microscopes and other instruments with high resolution to provide you with clear and high-quality pictures.

  • High standard

    The experimenters have many years of successful experiment experience and can guarantee the standard of experiment operation and experiment process.

  • Short time and low cost

    We provide customers with the most comprehensive services at the most favorable price and help customers improve the efficiency of research.

  • Safety

    All experiments have signed confidentiality agreements, focusing on protecting customer privacy.

If you are interested in our services, please contact us for more detailed information.

* For scientific research only

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