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IDENTIFICATION OF APOPTOTIC PROTEINS Emily Young Carr

Identification of Apoptotic Proteins

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Identification of Apoptotic Proteins. Emily Young Carr. Purpose. To determine if cells under tumor-like stress die by apoptosis. To test if Bcl-XL and Bax are involved when cells die under tumor-like stress. . Background Information. - PowerPoint PPT Presentation

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Page 1: Identification of Apoptotic Proteins

IDENTIFICATION OF APOPTOTIC PROTEINS

Emily Young Carr

Page 2: Identification of Apoptotic Proteins

PurposeTo determine if cells under tumor-like stress die by apoptosis.

To test if Bcl-XL and Bax are involved when cells die under tumor-like stress.

Page 3: Identification of Apoptotic Proteins

Background Information Apoptosis is a naturally occurring process in which a

no longer functional cell dies. (Campbell & Reece, 2008).

  The cell’s decision of whether to live or die occurs in

the mitochondrion (Campbell & Reece, 2008). The release of cytochrome c from the mitochondrion triggers apoptosis.(Weinberg, 2006).

  The protein Bcl-XL inhibits apoptosis by stopping the

passage of cytochrome c out of the mitochondrion (Adams & Cory, 1998).

 

Page 4: Identification of Apoptotic Proteins

Background Information cont. The protein Bax promotes apoptosis by heightening the

mitochondrial membrane’s permeability so cytochrome c can be released(Marzo, 1998).

  To identify a specific protein, size markers are used.

Bax is typically seen at about 21 kDa (Cell Signaling, 2011) however Bax is known to cleave during apoptosis to a molecular weight of 18 kDa (Cao, 2003).

Bcl-XL is located at about 28 kilodaltons (Cell Signaling, 2011).  Recent research has shown that cells under tumor-like stress die

due to lipid deprivation (Young, 2012).

Lipids rescue viability in cells under tumor-like conditions. Therefore the apoptotic process is halted by the presence of lipids in cells (Young, 2012).

Page 5: Identification of Apoptotic Proteins

The diagram illustrates the release of cytochrome c triggering the apoptotic cycle. The role of anti- and pro- apoptotic proteins

in this occurrence is also shown.

Page 6: Identification of Apoptotic Proteins

Hypothesis Cells under lipid deprivation die through

the mechanism of apoptosis. If both proteins are involved, then higher

levels of the pro-survival protein Bcl-XL will be found in both the replete and limited serum and oxygen with fatty acid supplementation conditions while higher levels of the pro-apoptotic protein Bax will be found in the limited serum and oxygen condition.

Page 7: Identification of Apoptotic Proteins

Procedure  1. The plates were set up in a Bio-Rad apparatus. 2. 20 mL of 15% acrylamide solution was prepared. 3. The gel was poured and the plates were sealed on top with isopropyl

alcohol.  4. 4 mL of stacking gel was prepared and added to the gels.  5. Combs were placed in the stacking gel to create wells.  6. The gels were given 1 hour to polymerize.  7. The protein extracts were prepared. There were 3 samples of each nutrient

condition.o 5x loading dye was added to the samples.o Samples were heated for 5 minutes at 95°C.

  8. The extracts were loaded onto the gels which were run for 2.5 hours.  

Page 8: Identification of Apoptotic Proteins

Procedure cont. 9. The proteins were transferred from the gels to nitrocellulose membranes

using a transfer apparatus.

10. Membranes were stained with Ponceau-S dye and then washed.

11. Membranes were blocked for an hour with 5% milk in TBST.

12. 10 mL of each primary antibody was prepared. The antibodies were hybridized to the membranes in the cold room overnight.

  13. Unbound antibodies were removed and the membranes were washed.  14. The secondary antibodies were placed on the membrane for an hour. Then

the secondary antibodies were removed and the membranes were washed.   15. ECL, a chemiluminescence reagent, was added to the membranes which

were taken to a dark room and developed.   16. For a control, the blots were stripped with a stripping buffer. Then the β-

actin antibody was added to the blots and the same procedure for detection which was previously described was performed.

Page 9: Identification of Apoptotic Proteins

This image depicts gel electrophoresis. Note the wells in the gel for protein loading.

This image is intended as a reference point and is not identical to this experiment’s procedure.

Diagrams

Page 10: Identification of Apoptotic Proteins

This image depicts protein transfer. This image is intended as a reference point and is not

identical to this experiment’s procedure.

Diagrams cont.

Page 11: Identification of Apoptotic Proteins

Photographs

1.5mm plates with polymerizing gels. Vaseline seal on bottom.

The gels with wells before protein loading.

Running the gels. Bubbles show current.

The nitrocellulose membranes between the first and second antibodies.

All photographs were taken by m

e.

Page 12: Identification of Apoptotic Proteins

Variables Independent Variables-

o the apoptotic protein being tested for, either Bcl-XL or Baxo the nutrient condition under which the cells of the extracts

were cultured

Dependent Variable- the expression of the proteins in the three nutrient conditions

Control- β actin was used as a loading control

Constants- the amounts of antibodies and solutions used, the materials used, temperatures, the location used for experimentation

Page 13: Identification of Apoptotic Proteins

Scans of Bcl-XL and Bax Blots

Scans show a western blot analysis of the expression of Bcl-XL and Bax under replete (R), serum and oxygen limitation (SO), and serum and oxygen limitation with fatty acid supplementation (SO+FA).

Page 14: Identification of Apoptotic Proteins

Tables for Bax

Group 1 Group 2 Group 3 Average

R 5039.6 25750.5 34252.2 21680.7667

SO 128628.7 71420.3 61212 87087

SO+FA 21073.7 19204.9 19193.1 19823.9

Average Standard Deviation T-Test Values

R 21680.7667 15025.5132 0.04496266

SO 87087 36336.4414  

SO+FA 19823.9 1082.37463 0.0327503

Levels of Cleaved Bax in Three Different Nutrient Conditions

Statistical Analysis for Bax

Page 15: Identification of Apoptotic Proteins

Tables for Bcl-XL

Column1 Group 1 Group 2 Group 3mn4 Averagelumn5

R 108307.21 68833.931 101843.825 92994.9887

SO 101734.169 101840.462 101834.938 101803.19

SO+FA 101851.131 101844.746 101851.495 101849.124

Average Standard Deviation

R 92994.9887 21172.1838

SO 101803.19 59.8374294

SO+FA 101849.124 3.09927809

Levels of Bcl-XL in Three Different Nutrient Conditions

Statistical Analysis for Bcl-XL

Page 16: Identification of Apoptotic Proteins

Bax Graph

R SO SO+FA0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000Levels of Cleaved Bax in Different Nutrient Conditions

Nutrient Condition

Rel

ativ

e In

tens

ity

*

Page 17: Identification of Apoptotic Proteins

R SO SO+FA0

25000

50000

75000

100000

125000 Levels of Bcl-XL in Different Nutrient Conditions

Nutrient Condition

Rel

ativ

e In

tens

ityBcl-XL Graph

Page 18: Identification of Apoptotic Proteins

Conclusion Hypothesis- The dual hypothesis was that…

Cells under lipid deprivation die through the mechanism of apoptosis. If both proteins are involved, then higher levels of the pro-survival protein

Bcl-XL will be found in both the replete and limited serum and oxygen with fatty acids conditions while higher levels of the pro-apoptosis protein Bax will be found in the limited serum and oxygen condition.

 It was found that cleaved Bax is involved in the lipid rescue phenotype and Bcl-XL is not. The involvement of Bax in the lipid rescue phenotype supports the idea of apoptosis as the death mechanism. Therefore, the hypothesis was supported.

Applications Increased knowledge of the phenotype this experiment focused on is

helpful in determining which areas of tumors to target when developing new cancer treatments.

The unexpected result which suggests that cleaved Bax is involved in the lipid rescue phenotype could help in the development of artificially cleaved Bax as a more effective initiator of apoptosis.

Page 19: Identification of Apoptotic Proteins

Conclusion cont. Possible Sources of Error

o Non-specific binding particularly of Bax antibodies may have made it appear as though Bax was involved when it was not.

o The occurs because antibodies bind to the nitrocellulose membrane instead of the proteins on the membrane.

o However, steps were taken to prevent this, the blocking of the membranes in 5% milk in TBST.

Improvements and Further Experimentationo Other members of the Bcl-2 family such as Bcl-2 could be

tested in a western blot with extracts of cells cultured under the same conditions used in this experiment.

o It would be interesting to determine which other pro-apoptotic proteins are involved in the lipid phenotype.

Page 20: Identification of Apoptotic Proteins

Acknowledgements

Thank you to my teacher Mrs. Giannantonio, Simon Lab at the Abramson Family Cancer Research Institute, and most of all to Dr. Gina Young.

Thank you to all sources used. Adams, J., & Cory, S. (1998, August 28). The Bcl-2 protein family: Arbiters of cell survival. Science, 281. Abstract retrieved from

http://www.ncbi.nlm.nih.gov/ pubmed/ 9735050 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (Eds.). (2002). Molecular biology of the cell (4th ed.). New York, NY: Garland

Science. (Original work published 1983) Bensaccount (2009, September 12). SDS-PAGE Electrophoresis [Digital Diagram]. Retrieved from Wikipedia database. Campell, N. A., Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Jackson, R. B. (Eds.). (2008). AP edition biology (8th

ed.). San Francisco, CA: Pearson Benjamin Cummings. Cao, X., Deng, X., & May, W. S. (2003, October 1). Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like

protease may rapidly degrade p18 Bax. Blood Journal, 102. Cell signaling technology. (2011). [Brochure]. Beverly, MA: New England BioLabs. DeBarardinis, R. J., Sayed, N., Ditsworth, D., & Thompson, C. B. (2008). Brick by brick: Metabolism and tumor cell growth. Current Opinion in

Genetics and Development, 18(1), 54-61. The Intrinsic Mitochondrial Apoptosis Pathway] [Digital Diagram]. (n.d.). Retrieved from AbD serotec database. Jürgensmeier, J., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., & Reed, J. (1998, April 28). Bax directly induces release of cytochrome c from

isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 95. Abstract retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/ 9560217

Key, J. P. (1997). MODULE s5 - “t” TEST [Math Resource]. Retrieved January 31, 2011, from Research Design in Occupational Education database.

Marzo, I., Brenner, C., Zamzami, N., Jürgensmeier, J., Susim, S., Vieira, H., . . . Xie, Z. (1998, September 25). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science, 281. Abstract retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/ 9748162

Pierce, R. (2011, September 12). Standard deviation [Math Resource]. Retrieved January 31, 2011, from Math is Fun - Maths Resources database.

Vander Heiden, M., Li, X., Gottleib, E., Hill, R., Thompson, C., & Colombini, M. (2001, June 1). Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. The Journal of biological chemistry, 276. Abstract retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/ 11259441

Weinberg, R. A. (Ed.). (2006). The biology of cancer. New York, NY: Garland Science. Young, R. M. (under revision). Dysregulated mTORC1 renders cells dependent on serum lipids for survival under tumor-like stress. Cell

Metabolism.

Thank you to my audience for listening. I will now take any questions you may have.