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Friday, January 15, 2010

Protein Purification Protocols (1)

Protein Purification and Identification Protocols

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity.

1. ALKYL Aspartamide HIC Columns

2. Comparison of affinity tags for protein purification.

3. DEAE Column Preparation

4. DEAE column: FPLC

5. Green Fluorescent Protein Isolation (Gary J. Lindquester)

6. GST Fusion Protein Preparation

7. GST Fusion Protein Purification from Yeast

8. GST-Fusion Protein Purification

9. HeLa Cell Nuclei Preparation (John Garland, The Biochemistry and Molecular Biology Department of The James H. Quillen College of Medicine in Johnson City, TN)

10. HILIC and SEC Columns PolySULFOETHYL and PolyHYDROXYETHYL Columns

11. Isolation of Protein from Tissue (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)

12. Making Affinity Columns

13. Making Conductivity Curves (Waters Lab)

14. PI-PLC Purification (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

15. Polyaspartic Acid Weak Cation Exchange Columns

16. PolyGLYCOPLEX Complex Carbohydrates Columns

17. PolySULFOETHYL Aspartamide Strong Cation Exchange Columns

18. PolyWAX LP Weak Anion Exchange Columns

19. Preparation of Tonsil Lysate (Springer Lab, Harvard University)

20. Protein Extraction (Frank Bottone Jr.)

21. Protein Extraction from Tissues (Reddy research laboratory, Neurological Sciences Institute, Oregon Health & Science University)
Protein Isolation Protocols (Lamond Lab, University of Dundee)

22. Protein Isolation using Trizol (Frank Bottone Jr.)

23. Protein Purification

24. Protein Purification

25. Protein Purification

26. Protein Purification

27. Protein Purification (handbook)

28. Protein Purification (Michael Blaber)

29. Protein Purification (Michael Blaber)

30. Protein Purification (Michael Blaber)

31. protein purification (Pictures)

32. Protein Purification and Analysis Protocols and Applications Guide (Promega)

33. Protein Purification on 1G3 Column (Bjorkman Group, Howard Hughes Medical

34. Protein Purification Protocols (The Protein Expression and Purification Facility, EMBL)

35. Protein Sample Preparation & Protein Purification (Invitrogen)

36. Purificaiton of Protein by Affinity Chromatography (Krause Lab)

37. Purification of GST Fused Proteins

38. Purification of GST Fused Proteins

39. Purification of His-tagged proteins from Sf9 cells under native conditions

40. Purification of Proteins (NCBI)

41. Purification Scheme for Ubiquitin Expression Lysate (Sosnick Lab, University of Chicago)

42. Purification of Maltose Binding Proteins Fused Proteins

43. Purification of MBP (maLTose-Binding Proteins) Fused Proteins (Bowtell Lab

44. Removal of Nucleic Acids from Protein (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

45. RNA and protein extraction from the same tissue (Ivan J Delgado)

46. SDS Removal Guard Columns

47. Small Scale MBP-fusion Protein Purification (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

48. Subcellular Fractionation of Proteins (Bowtell Lab Manual, Peter MacCallum Cancer Institute)

49. Sucrose Density Gradient Fractionation of Yeast Membranes (Dohlman Lab)
Test Tube for Ion Exchange Chromatography (Dr. Mario Lebendiker, The Hebrew University of Jerusalem)

50. The Protein Purification Facility (Dr. Mario Lebendiker)

51. Tips for acid elution of peptides (Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)

52. Whole Cell Protein Extracts (Morrisey Lab, University of Pennsylvania)
This is for both cytoplasmic and nuclear proteins.
Protein Purification - How to Purify Proteins.

Suggested Reading

Protein Purification HandbookWhat is Biotechnology?Protein Engineering MethodsWhat is a Protein?What are Enzymes?

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Stem Cell Research Progresses

Stem Cells

Stem Cells Might Reverse Heart Damage from Chemo (12/30/2009, HealthDay)

Stem Cell Research Gets a Boost from Vitamin C (12/24/2009, HealthDay)

Most Stem Cells Used in Research Come from Whites (12/17/2009, HealthDay)

Adult Stem Cells Can Repair Damaged Heart (12/04/2009, HealthDay)

First Human Embryonic Stem Cell Lines Approved for Use Under New NIH Guidelines (12/02/2009, National Institutes of Health)

U.S. Approves New Stem Cell Lines for Publicly Funded Research (12/02/2009, HealthDay)

New 'Skin' from Stem Cells (11/19/2009, HealthDay)

Lifelong Exercise Keeps Seniors Young at Heart (11/18/2009, HealthDay)

Stem Cells May Improve Heart Bypass Results (11/17/2009, HealthDay)

Stem Cells May Offer Alternative to Lung Transplants (11/06/2009, HealthDay)

Stem Cells Repair Acute Lung Injury in Mice (11/05/2009, HealthDay)

NIH-Funded Researchers Transform Embryonic Stem Cells Into Human Germ Cells (10/28/2009, National Institute of Child Health and Human Development)

Stem Cell Transplants Stalled Blindness in Rats (10/19/2009, Reuters Health)

Transplant May Treat Metabolic Disorders in Womb (10/16/2009, HealthDay)

Stem Cells Grow Heart Tissue in Lab (10/15/2009, HealthDay)

Stem Cells Being Tested to Fight Pancreatic Cancer (10/14/2009, HealthDay)

Lab Study Makes Strides in Mending Hearts (10/12/2009, HealthDay)

New Way to Make Stem Cells? (10/08/2009, HealthDay)

Stem Cell Research Offers Hope for Colon Cancer Vaccine (10/08/2009, HealthDay)

Stem Cells: Online Resource (2)

News Sources

Biotechnology Industry Organization Up-to-date news on stem cells, cloning, and regenerative medicine.

EurekAlert! A science news site provided by the American Association for the Advancement of Sciences.

Forum on Science Ethics & Policy Links to informational, legislative, editorial, and other sites discussing stem cell research.

National Geographic: The Power to Divide Feature story on the science and politics of stem cell research.

Nature: Focus on Stem Cells A selection of articles and features on stem cells, including the specially commissioned Stem Cells Insight.

NewScientist: Cloning and Stem Cells A monthly update on stem cell technology.

The New York Times: Stem Cells A free collection of articles about stem cells published in The New York Times.

NOVA scienceNOW: Stem Cells Update From the PBS series (originally aired April 19, 2005). Includes dispatches on the politics of stem cells, the cloning process, and related science news.

PBS: Miracle Cell This episode of the series Innovation explores the use of stem cells for regenerative therapies. (Originally aired April 13, 2004)

Scientific American Provides news coverage on stem cell research. See The Stem Cell Challenge, a feature article from the June 2004 issue.

SciTech Daily Review Offers a range of news articles, features, and commentaries about science and technology topics.

Stem Cell Research News An online newsletter that features stories about stem cells of all types.

Stem Cells The International Journal of Cell Differentiation and Proliferation.

Time: The Stem Cell Debate Stories and resource links on stem cell news.

The Washington Post: Stem Cell Research A collection of articles on stem cell research. (Free registration required to search for other articles within the previous two weeks; fee required to search for older archived articles.)

News Web Logs

The Niche A blog hosted by Nature Reports Stem Cells that offers a forum for debate and commentary on stem cell research and its wider implications for ethics, policy, business, and medicine.

The Stem Cell Blog Discussion on the science, ethics, business, and politics of stem cell research.

Stem Cell News Abstracts of current news articles regarding stem cells, with links to sources.

Stem Cell Research Progress Blog—Information About Stem Cells Discussion of stem cell news and events.

Professional Associations

American Association for the Advancement of Science (AAAS) The AAAS policy brief on stem cell research.

American Medical Association (AMA) Provides information on the basics of stem cell research and highlights relevant policy developments.

American Society for Cell Biology (ASCB) Facilitates the exchange of scientific knowledge about cell biology by disseminating research and training students and young investigators.

American Society for Reproductive Medicine (ASRM) FAQs about cloning, stem cell research, and ASRM's position on the issues.

Association of American Medical Colleges (AAMC) Discussion of stem cell research.

Federation of American Societies for Experimental Biology (FASEB) Seeks to help biomedical and life scientists improve people's health, well-being, and productivity.

The International Cord Blood Society (ICBS) Dedicated to the advancement of stem cell research with an emphasis on umbilical cord blood stem cells.

International Society for Stem Cell Research (ISSCR) Works to exchange information on stem cells, encourage research involving stem cells, and educate the public in all areas of stem cell research and application.

Related Federal Government Sites

FDAA's Center for Biologics Evaluation and Research Determines whether stem cells are safe to use in clinical trials.

Health Resources and Services Administration's The C.W. Bill Young Cell Transplantation Program Provides information and support to patients who need a potentially life-saving bone marrow transplant or umbilicial cord blood transplant.

NIDDK Investigator's Guide to Human Embryonic Stem Cell Research Provides information for researchers supported by the National Institute of Diabetes & Digestive & Kidney Diseases.

NIH Bioethics Resources on the Web Contains a broad collage of annotated web links that provide background information and various positions on issues in bioethics.

The President's Council on Bioethics Advises the President on ethical issues related to advances in biomedical science and technology, including stem cells.

Research Programs at Universities and Institutions

Harvard Stem Cell Institute Supports research into all aspects of stem cell biology, with special emphasis on those areas with the greatest potential for improving human health.

McGowan Institute for Regenerative Medicine Established for University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center scientists and clinical faculty working to develop tissue engineering, cellular therapies, biosurgery, and artificial and biohybrid organ devices.

National Human Neural Stem Cell Resource Provides neural stem cells harvested from the post-natal, post-mortem, human brain to the research community for stem cell research.

The New York Stem Cell Foundation (NYSCF) Offers grants, fellowships, and symposia for stem cell researchers; educates the public about stem cells; and establishes collaborative stem cell research facilities.

Pittsburgh Development Center of Magee-Womens Research Institute Explores the molecular biology of cell function, including the potential of stem cells for treating human disease.

Sloan-Kettering Institute Part of the Memorial Sloan-Kettering Cancer Center, the world's oldest and largest private institution devoted to patient care, education, and research into cancer.

Stanford University School of Medicine/Institute for Cancer/Stem Cell Biology and Medicine Explains Stanford's involvement and perspective on stem cell issues, with links to related sites.

Texas A&M University Mesenchymal Stem Cell Distribution Prepares and distributes well-characterized marrow stromal cells (MSCs) derived from adult human and rodent bone marrow using standardized protocols.

Texas Heart Institute Stem Cell Center Dedicated to the study of adult stem cells and their role in treating cardiovascular disease, including clinical trials (in human patients), as well as many preclinical studies (in the laboratory) using stem cells.

University of California, San Francisco/Developmental and Stem Cell Biology Program Highlights of UCSF human embryonic stem cell research.

University of Massachusetts: International Stem Cell Registry The Potential of Human Pluripotent Stem Cells

University of Minnesota: Stem Cell Institute Works to enhance understanding of stem cells' potential to improve human and animal health.

University of Wisconsin/Embryonic Stem Cell Research Scientists at UW-Madison were the first to successfully isolate and culture human embryonic stem cells.

State Initiatives for Stem Cell Research

California Institute for Regenerative Medicine (CIRM) Makes grants and provides loans for stem cell research, research facilities, and other research opportunities at California universities and research institutions.

Connecticut Stem Cell Research Program Provides grants-in-aid for conducting embryonic or human adult stem cell research.

Illinois Regenerative Medicine Institute (IRMI) Awards grants to Illinois medical research facilities to fund stem cell research involving adult, cord blood, and embryonic stem cells.

Maryland Technology Development Corporation (TEDCO) Provides emerging technology companies and university researchers with funding and specialized technical assistance.

New Jersey Stem Cell Research Program Awards individual research and core facility grants to support human embryonic stem cell research.

New York Stem Cell Science (NYSTEM) Supports basic, applied, translational or other research and development activities that will advance scientific discoveries in fields related to stem cell biology.

Ohio Center for Stem Cell and Regenerative Medicine A multi-institutional center (Case Western Reserve University, University Hospitals Case Medical Center, the Cleveland Clinic, Athersys, Inc., and Ohio State University). Provides basic and clinical research programs, biomedical and tissue engineering programs, and the development and administration of new therapies.

Stem Cell Research State Laws The National Conference of State Legislatures provides an overview of the state laws and pending legislation on stem cell research.

Interstate Alliance on Stem Cell Research (IASCR) Fosters effective interstate collaboration, helps states develop research programs, and promotes efficient and responsible use of public funds.

Stem Cells: Online Resource (1)

Online Resources

The links included here may connect you to other Internet sites that operate independently of the NIH. The NIH is not responsible for the availability or content of other sites. Permission to reproduce information at other sites may be required. The NIH does not endorse, warrant, or guarantee the information, services, or products described or offered at these external sites.

Annotated Bibliography

Cellular Therapy: Potential Treatment for Heart Disease, Food and Drug Administration (FDA), 2004.
Despite advances in treatment, ischemic heart disease and congestive heart failure are major causes of death in the United States. Cell therapies for treating these diseases are of interest to medical researchers. This resource outlines the issues surrounding clinical studies of human stem cells and the FDA's role in ensuring safe studies.

Stem Cell Therapy for Heart Patients, National Public Radio Talk of the Nation Audio, April 2004.
This one-hour audio program discusses injecting stem cells into heart patients to improve blood flow. This new treatment for heart disease uses the patient's own bone marrow to restore heart function.

Reaping the Benefits of Genomic and Proteomic Research: Intellectual Property Rights, Innovation, and Public Health, Committee on Intellectual Property Rights in Genomic and Protein Research and Innovation, National Research Council, 2005.
The National Academy of Sciences reports on the granting and licensing of intellectual property rights on discoveries relating to genetics and proteomics and the effects of these practices on research and innovation.

Stem Cells and the Future of Regenerative Medicine, by Commission on Life Sciences, 2002.
Stem Cells and the Future of Regenerative Medicine summarizes what we know about adult and embryonic stem cells. It also provides an overview of the moral and ethical problems that arise from the use of embryonic stem cells, compares the likely impact of public and private research funding on progress in the field, and discusses approaches to appropriate research oversight. Based on the insights of leading scientists, ethicists, and other authorities, the authors make recommendations regarding the use of existing stem cell lines versus new lines in research, the important role of the federal Related Federal Government Sites in this field of research, and other fundamental issues impacting potential stem cell-based therapies.

Unlocking the Promise of Stem Cells, Harvard Stem Cell Institute, March 2004.
View an interactive videoconference in which University researchers discuss the Harvard Stem Cell Institute, created to move cutting-edge research on embryonic stem cells from the lab to the clinic.

Beyond Therapy: Biotechnology and the Pursuit of Happiness, Report from Former President Bush's Council on Bioethics, 2003.
Can biotechnology satisfy our human desires—for better children, superior performance, ageless bodies, and happy souls? This report from the President's Council on Bioethics says these possibilities present us with profound ethical challenges and choices. Not declaring "findings," but holding an inquiry—inviting us all to think and debate—the President's Council sought the ideas of dozens of celebrated scientists, thinkers and writers, including such Council members as Francis Fukuyama, Charles Krauthammer, Michael Sandel, and James Q. Wilson, as well as witnesses Steven Pinker, Daniel Schacter, Lawrence Diller, Steven Austad, and S. Jay Olshansky.

Directories and Databases

MEDLINEplus—Stem Cells/Stem Cell Transplantation A consumer health database that includes news, health resources, clinical trials, and more.

Nature Biotechnology Directory A directory of companies engaged in biotechnology research.

PubMed Contains references to literature from more than 4,300 life sciences journals.

Stem Cell Database A joint project of Princeton University and the University of Pennsylvania that contains data on the molecular phenotype of hematopoietic stem cells.

Disease-Specific Organizations and Advocacy Groups

Adult Stem Cell Research Network An internet-based project of The Cell Therapy Foundation designed to be a well-maintained and reliable source of information for the public regarding adult stem cell research, as well as to be a community of practice and collaboration among fellow researchers.

Alliance for Aging Research This citizen advocacy organization works to improve the health and independence of Americans as they age.

ALS Association (ALSA) Dedicated to the fight against Amyotrophic Lateral Sclerosis (also known as Lou Gehrig's Disease), ALSA offers this primer on stem cells.

American Cancer Society Offers information on stem cell transplantation for children with leukemia.

Arthritis Foundation Discusses whether a stem cell transplant cures arthritis.

Christopher and Dana Reeve Foundation Funds research on treatments for central nervous system disorders and works to improve the lives of people with disabilities through a grants program, paralysis resource center, and advocacy efforts.

Coalition for the Advancement of Medical Research Advocates the advancement of breakthrough research and technologies in regenerative medicine, including stem cell research and somatic cell nuclear transfer.

Juvenile Diabetes Research Foundation Using stem cells to restore normal blood sugar, plus information on recent research and policy.

Lymphoma Information Network A page on bone marrow and stem cell transplants.

March of Dimes Aims to give babies a fighting chance against such threats to their health as prematurity, birth defects, and low birthweight.

Research!America Frequently Asked Questions (FAQs) on stem cell research.

Michael J. Fox Foundation for Parkinson's Research Dedicated to ensuring the development of a cure for Parkinson's disease through an aggressively funded research agenda.

National Health Council (NHC) Works to promote the health of all people by advancing the voluntary health movement.

National Marrow Donor Program® (NMDP) Maintains an international registry of donors for all sources of blood stem cells used in transplantation: bone marrow, peripheral blood, and umbilical cord blood.

Spinal Cord Injury Information Network Links to articles about stem cells, particularly for spinal cord injuries.

Educational Resources

Stem Book A collection of invited, original, peer-reviewed chapters on stem cell biology written by top researchers in the field at the Harvard Stem Cell Institute and worldwide. Stem Book is aimed at stem cell and non-specialist researchers.

ExploreStemCells A UK resource for the general public that discusses the use of stem cells in medical treatments and therapies.

How Embryonic Stem Cell Lines Are Made An online animation from the Dolan DNA Learning Center, Cold Spring Harbor Laboratory. (Requires Adobe Flash Player)

Human Embryonic Stem Cells An animated tutorial for the general public. (Requires Adobe Flash Player)

The National Academies Publications on stem cells, including Understanding Stem Cells: An Overview of the Science and Issues from the National Academies (2006), Guidelines for Human Embryonic Stem Cell Research (2005), Stem Cells and the Future of Regenerative Medicine (2002), and Scientific and Medical Aspects of Human Reproductive Cloning (2002).

NIH-Supported Science Education Partnership Award (SEPA) Projects

Cellular Universe: The Promise of Stem Cells Educates visitors about advances in cell biology and stem cells so they can make more informed health-related decisions, explore new career options, and better understand the role of scientific research in healthcare.

If a Starfish Can Grow a New Arm, Why Can't I? This Pittsburgh Tissue Engineering Initiative (PTEI) educational program informs middle school students, their teachers, and the general public about tissue engineering and its applications.

Regenerative Medicine Partnership in Education Duquesne University's SEPA project offers planetarium shows, movies, and educational Web tools.

NWABR Stem Cell Curriculum This 6-lesson unit explores the scientific and ethical issues involved in stem cell research. It features hands-on, engaging classroom activities that have been showcased in the journal American Biology Teacher.

Life Science Learning Center The University of Rochester educational program provide a general overview of stem cell biology and an introduction to stem cell research. Extended Version Stem Cell Biology module features a series of 10 short lessons on stem cells with hands-on lab activities and animated slide show. The Short Version” of the Stem Cell Biology module can be completed in two 40-minute class periods. Instructions for setting up the lab activity are included in the teacher guide.

A Stem Cell Story This 15-minute movie from EuroStemCell introduces the world of stem cell research and is available as a video podcast.

Stem cells: Cells with Potential Part of the San Francisco Exploratorium's Microscope Imaging Station.

Stem Cells: Engage A high school teaching resource on stem cell research developed in Canada.

Stem Cells in the Spotlight and Cloning In Focus The Genetic Science Learning Center at the University of Utah presents these outreach education programs for high school and undergraduate students and teachers.

Stem Cell ResourcesSM A resource for the educational community on stem cells.

Tissue Engineering Planetarium Show Online movies about the body's ability to heal itself.

Tissues of Life: Stem Cells An interactive comic explaining where stem cells are found in the body and how they are gathered.

World Stem Cell Map Reflects national policy and whether public funds may be used to pursue stem cell research using IVF embryos donated from fertility clinics.

International Research

Australian Stem Cell Centre Unites leading Australian academic researchers with the biotechnology industry to develop innovative therapeutic products.

Canadian Institutes of Health Research (CIHR) CIHR is the major federal agency responsible for funding health research in Canada. The site offers links to stem cell research guidelines, the Stem Cell Oversight Committee, and funding opportunities.

Centre for Stem Cell Biology (UK) Part of the University of Sheffield.

EuroStemCell Eleven academic institutes and enterprises from eight European countries compare stem cell information and evaluate their therapeutic potential.

The Goldyne Savad Institute of Gene Therapy (Hadassah University Hospital, Israel) Information on the Institute's Research Group on human embryonic stem cells.

Health Insite (Australia) List of stem cell resources.

Human Embryonic Stem Cell Research in China A September 2002 Report from the U.S. Embassy, Beijing.

Institute for Stem Cell Research (UK) Includes links to its research groups and collaborative initiatives.

Institute of Human Genetics at the University of Newcastle upon Tyne Outlines their current research projects.

The International Stem Cell Forum (ISCF) Encourages international collaboration and funding support for stem cell research.

Kyoto University (Japan) Home page for the University's Stem Cell Research Center

RIKEN Center for Developmental Biology (Japan) Home page for the Center's Laboratory for Stem Cell Biology

Scottish Stem Cell Network A multidisciplinary forum of scientists and clinicians advancing stem cell biology into identifiable health benefits.

Stem Cell Society Singapore Serving the stem cell research community of Singapore.

Stem Cells: Therapies for the Future? (EU) News and information about this 2001 European Union conference.

Tekes—National Technology Agency of Finland Tekes is the main public funding organization for research and development in Finland.

UK Medical Research Council Information on the MRC's stem cell initiative.

The UK Stem Cell Bank Information about stem cell lines available from the UK bank.

UK National Stem Cell Network Promotes research activities and events at the national level to speed the translation of basic stem cell research into therapeutic applications in the control of degenerative diseases.