Cells under Microscope

Chem 110L

Introductory Biochemistry Laboratory

Chloroplast

 

Lecturer:

Dr. Kalju Kahn
Office: 2623 PSB-N
Office hours: Tuesday 10:00-11:00 PM and by appointment
Phone: 893-6157
E-mail: kalju@chem.ucsb.edu
Web site: http://www.chem.ucsb.edu/~kalju

Teaching Assistants


Section 1 (MW 6-9:50):		Abril Estrada			aestrada@chem.ucsb.edu
	Office hours: TBA			

Section 2 (TR 2-5:50):		Jeanine Boucher			boucher@chem.ucsb.edu	
	Office hours: TBA

Section 3 (TR 6-9:50):		Richard Chapleau		rchapleau@chem.ucsb.edu
	Office hours: TBA 		    	
    	

Mission statement

The purpose of Chem 110L is to offer hands-on experience with modern methods of separation, identification, and characterization of biomolecules. The course will strengthen your understanding of material taught in Chem 142A (Biochemistry Lecture). In Chem 110L, you will do experiments with biomolecules such as nucleic acids, proteins, sugars, and lipids. The 1 hour lecture series focuses on principles behind each experiment, and explains instrumental techniques and methods that you will use to accomplish your goals.

Schedule for Fall 2008


	Lecture:        Mon 3:00-3:50   Place:  Phelps 1260
	
	Lab section 1:  Mon 6:00-9:50;  Wed 6:00-9:50;
	Lab section 2:  Tue 2:00-5:50;  Thu 2:00-5:50;
	Lab section 3:  Tue 6:00-9:50;  Thu 6:00-9:50;
	
	  
	Lab sections are in PSB-N 2619 unless otherwise noted
Syllabus General information about the course. PDF
Schedule Schedule MW6 PDF
Schedule Schedule for TR2 PDF
Schedule Schedule for TR6 PDF
Lab Text Required Theory Manual PDF
Textbook Recommended for students who are planning to take also 125L and 112L Link
Last Year Course materials from Fall 2007 Link
Exam Preparation for the exam: topic list PDF
Exam Preparation for the exam: Sample Questions PDF
Exam Preparation for the exam: Year 2002 Final PDF

Experiments

Students in the class do not have to purchase the laboratory manual. Each chapter of the lab manual can be downloaded here in the PDF format. Please note that you can follow hyperlinks that are in the PDF files by clicking on the link. Links to external literature sources are given later below.

Experiments Download Adobe Acrobat Here Acrobat
Lab 1: Macromolecular visualization Tutorial
Lab 2: Operations Manual: molar absorbtivity of urate PDF
Lab 2: Operations Manual: Mathematica Tutorial PDF
Lab 3: Agarose gel electrophoresis of DNA isoforms PDF
Lab 4: Thermal denaturation of double-stranded DNA PDF
Lab 5: Optical microscopy PDF
Lab 6: Identification of cold-induced proteins PDF
Lab 7: Quantitative enzymatic determination of glucose PDF
Lab 8: Identification of saccharides present in foodstuff by TLC PDF
Exp 9: Determination of the iodine value of a lipid by 13C NMR PDF
Exp 10: Light-induced proton gradient in chloroplast PDF

Literature Required or optional reading in PDF Acrobat
General Fitting Models to Biological Data (advanced) PDF
Exp 3: Microscopy: Dr. Matsumoto's Presentation Slides (2005) PDF
Exp 3: Microscopy: Perspective Story "How to Build a Superlens" by David R. Smith PDF
Exp 3: Microscopy: Fang et. al.: "Sub-Diffraction-Limited Optical Imaging with a Silver Superlens " PDF
Exp 4: Thermal denaturation of double-stranded DNA (advanced) PDF
Exp 5-1: Proteomics: Sample Preparation PDF
Exp 5-1: Proteomics: Plant Proteome Analysis by MS (Newton, 2004) PDF
Exp 5-1: Proteomics: Plant Cold Acclimation? (Thomashow, 2001) PDF
Exp 5-1: Proteomics: Cold Stress Responses (Shinozaki, 2003) PDF
Exp 5-2: Electrophoresis: DryStrip Kit Manual PDF
Exp 5-2: Electrophoresis: Power Supply Manual PDF
Exp 5-2: Proteomics: Isoelectric Focussing PDF
Exp 5-3: Proteomics: Protein Electrophoresis, SDS-PAGE PDF
Exp 5-4: Proteomics: Two-dimensional Gel Electrophoresis in Proteomics PDF
Exp 9: 13C NMR (Lipids): Mercury 200 NMR Manual PDF
Exp 10: Photosynthesis: Colloquium Paper: Hu et al, PNAS 1998 PDF
Exp 9: 13C NMR (Lipids): Lecture Slides PDF
Review: How to prepare for the final (exam topics) PDF
Review: Sample exam (Chem 110L, 2002) PDF

Student Files

Lab Computer Mirror

Student files on the C:\Students\ disk on the computer are mirrored in the MW6, TR2, and TR6directories.

Microscopy Images

These images of bovine pulmonary arterial epithelial cells were recorded using a fluorescence microscope during visit to UCSB's Microscopy Facility. On the right is a color image obtained by combining three individual images, each showing one component of the cell. Below are three images of the same object, taken with a fluorescence microscope at three different wavelengths. An appropriate combination of these three files will give the color image of the cell, similar to the one shown on the right. You can click on each image to download the high-resolution file.

COMBINED COLOR IMAGE
Nuclei Microtubules Actin
Nuclei Microtubules Actin filaments

Below is a similar set of images from the year 2008. Notice that this time a different light or filter setting was used when the recording microtubules such that DAPI fluorescence also shows in this image. Notice the limited depth of field effect in microtubules image where the top right corder is very sharp but the bottom left corner is out of focus. Also notice problems with the actin image, presumably due to overly agressive postprocessing. In this case, three different color images were created to emphasize each of the components in the presence of other two.

Nuclei Microtubules Actin
Nuclei Microtubules Actin filaments

The images below are of human cheek epithelial cells as seen through the Olympus Provis microscope in three modes: brightfield, Nomarski interference contrast, and darkfield. The spherical structure seen in the center of the cell in brightfield and Nomarski image is the nucleus; the bright dots throughout the cell in the darkfield image are various granules. While the Nomarski image may appear more real a first sight, the dark and bright areas surrounding the edges of the nucleus and granules are artifacts of this imaging technique.

Brightfield Nomarski Darkfield
CHEEK CELLS CHEEK CELLS CHEEK CELLS

The images below show how fluorescence imaging can be used to visualize specific sub-cellular structures. The first image shows microtubules stained with a fluorescent dye; the image was recorded in black and white. The second image shows nuclei stained with a fluorescent compound DAPI; this image was also recorded in black and white but was obtained using excitation light of different wavelength than the microtubule image. To obtain the last image, separate colors were assigned to the two previous images before combining them into one.

Microtubules Nuclei Combination
Microtubules Nuclei Combo

The images below show how choice of filters allows to improve contrast in brightfield microscopy. The three images show a tissue slice with several cells underging mitosis. The DNA in cells organizes into chromosomes at early stages of mitosis (cell in upper-central part) and the two sets of daugther chromosomes separate in the early anaphase (cells in the lower-central part). The cells are treated with a red dye that binds to DNA. A red dye appears red to our eye in the brightfield microscope because it strongly absorbs blue and green light. The first image shows an unfiltered color view of the tissue. The next two images are recorded in black and white using two different color filters. Notice the improved contrast with an appropriate choice of the filter in the last image.

Unfiltered Red Filter Green Filter
Unfiltered Color Red Filter Green Filter

Proteomics: Gel Images

One of the projects in Chem 110L involves "discovery" of cold-induced proteins in ivy. Students will identify proteins that show either up- or down-regulation in expression upon cold-treatment. The identification is based on the comparison of the protein composition of normal and cold-stressed plants using 2D electrophoresis. This technique separates proteins first according to their isoelectric point values, and then, in a perpendicular dimension, according to the molecular weight.

Two different isoelectric focusing strips (pH 4-7 and pH 3-10NL) were used by students in different sections. The file here shows the pH versus distance profiles in these strips. You may find this reference map of Arabidopsis proteins helpful for analysis of proteins in ivy. Keep in mind that these are different plants, but in general most plant proteins tend to be similar to each other, especially in moleculr weight. If you have trouble comparing the two gels, check out this visual guide.


Proteomics Plant pH 4-7 IEF Plant pH 3-10 NL IEF
Ivy Gels '08

2D Gel: Regular lab 2D Gel: Make-up
Proteomics Warm Plant pH 3-10 NL IEF Cold Plant pH 3-10 NL IEF
Ivy Gels '07
100 µL of ivy extract + 25 µL of rehydration buffer (total 50 µg protein/gel)
2D Gel: Normal plant 2D Gel: Cold-treated plant
Proteomics Warm Plant pH 3-10 NL IEF Cold Plant pH 3-10 NL IEF
Ivy Gels '07
50 µL of ivy extract + 75 µL of rehydration buffer (total 25 µg protein/gel)
2D Gel: Normal plant 2D Gel: Cold-treated plant
Proteomics Warm Plant pH 4-7 IEF Cold Plant pH 4-7 IEF
Ivy Gels '06
2D Gel: Normal plant 2D Gel: Cold-treated plant

Proteomics Warm Plant pH 4-7 IEF Cold Plant pH 4-7 IEF
Arabidopsis 2004

2D Gel: Warm (control) plant 2D Gel: Cold-treated plant

Biochemistry Links

Protein Data Bank
PyMOL Molecular Visualization Program
Biomolecular Visualization Examples with PyMol.
Chemscape Chime to view 3D structures online.
How To View Stereo Pair Photographs
Stereo Viewing
Hints for studying biochemistry

UCSB links

UCSB General Catalog
UCSB Campus Map
UCSB Gold Login
UCSB Umail Access
Electronic Journals
UCSB Environmental Health and Safety
UCSB Rape Prevention Education Program


Course materials by Dr. Kalju Kahn, Department of Chemistry and Biochemistry, UC Santa Barbara. ©2003-2007