Isolation of Casein from Milk

  Isolation and Identification of Casein From Milk Course Notes

Milk is the probably the most nutritionally complete food found in nature. Whole milk contains vitamins (principally thiamine,riboflavin, panthothenic acid and vitamins A, B₁₂ and D), minerals (calcium, sodium, phosphorus, potassium, and trace minerals), proteins (which include all the essential amino acids), carbohydrates (mostly lactose), and lipids (fats). Whole milk is an oil in water emulsion, containing approximately 4% fat dispersed as very small (micron sized) globules. The fat emulsion is stabilized by complex phospholipids and proteins that are absorbed on the surface of the emulsion. Since the fat in milk is so finely dispersed it is more easily digested than fats from any other source.

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Isolation of Casein, Lactose, and Albumin from Milk

Milk is a food of exceptional interest. Not only is milk an excellent food for the very young, but humans 

have also adapted milk, specifically cow’s milk, as a food substance for persons of all ages. Many 

specialized milk products like cheese, yogurt, butter, and ice cream are staples of our diet.

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Experiment 11: Isolation and Characterization of Casein from Milk

Adapted from Experiment 21, “Isolation of Protein, Carbohydrate and Fat from Milk”, in

Mohr. S.C., Griffin, S.F., and Gensler, W. J. Laboratory Manual for Fundamentals of

Organic and Biological Chemistry by John McMurry and Mary E. Castellion,: nglewood

Cliffs, Prentice-Hall, 1994 and Wayne P. Anderson (4/2002)

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ISOLATION OF CASEIN FROM MILK

Purpose: In this lab you will be isolating the proteins casein and lactalbumin from a sample of milk. You

will use these values to determine the percent protein in milk compared to the listed value on the box.

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Isolation of Bacteria from the Environment - References

Isolation of bacteria from the environment http://delrio.dcccd.edu/jreynolds/microbiology/2420/files/bacteria_ubiquity.pdf

Laboratory Notes for BIO 1003 John H. Wahlert & Mary Jean Holland Examination of dental bacteria http://faculty.baruch.cuny.edu/jwahlert/bio1003/eubacteria.html

Culture and isolation of phototrophic sulfur bacteria from the marine environment http://adsabs.harvard.edu/abs/1970HWM....20....6T

Isolation of Bacteria from Soil Samples

ISOLATION OF SOIL BACTERIA: VIABLE TITER and PURE CULTURE
http://www2.fiu.edu/~makemson/MCB2000Lab/Exp4SoilBact.pdf

Isolation of an Unknown Bacterium from Soil
Department of Biological Sciences
University of Nevada, Las Vegas
http://www.ableweb.org/volumes/vol-14/6-steubing.pdf


ISOLATION OF PHOSPHATE SOLUBILISING BACTERIA FROM SOIL AND ITS ACTIVITY
http://biotechindia.files.wordpress.com/2007/12/isolation.pdf

Isolation of Soil Microorganisms
http://apps.caes.uga.edu/sbof/main/lessonPlan/microorganismIsolation.pdf


Isolation of bacteria from mechanic workshops’ soil environment contaminated with used engine oil
Department of Medical Laboratory Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Nigeria.


Isolation of soil Streptomyces as source antibiotics active against antibiotic-resistant bacteria
http://ejobios.com/pdf/ejob-8-11-2,9,73-82.pdf


ISOLATION OF SOIL BACTERIA FOR BIOREMEDIATION OF HYDROCARBON CONTAMINATION
http://www.chem.msu.su/rus/vmgu/031/88.pdf

RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols
http://www.engr.colostate.edu/~apruden/classes/ce742/Readings/Articles/RNAextractTechniques.pdf

LARGE SCALE PLASMID PREP CsCl METHOD

Triton lysis / CsCl method

Grow 500ml cultures with antibiotic selection shaking at 37oC.
When OD600 = 0.8 you may add chloroamphenicol to 125ug/ml.
Let it go overnight.

Spin down the cultures in sterile bottles at 5000rpm for 10 min at 4oC.
Make sure the seals on the bottle are properly seated and
the bottles are balanced within 0.1g.
Carefully decant the supernatant down the drain.
*You may replace the cap and store the pellet at -20oC.
*Before going on with the prep, be sure the ultracentrifuge and rotor are available
and sign up to use them.
You'll do one 30 min spin and one for 36 hours!

Prepare lysozyme on put on ice.
You'll need 0.5ml per culture.
Use plastic tube, and don't vortex.
For 1ml: 0.75ml water
0.25ml 1M Tris-Cl, pH 7-8
10mg lysozyme (stored in desiccator at -20oC)

If the pellet isn't soft , vortex it for a long time until it is.

Add to pellet 3ml 25% sucrose, 50mM Tris-Cl. Mix
Add 0.5ml lysozyme. Mix gently. Leave on ice for 5 min.

Add 1ml 0.5M EDTA, leave on ice for 5 min.

Add 4.5ml Triton juice. Mix gently.
[Triton juice: 4ml 10% Triton-X100, 20ml 0.5M EDTA, 20ml 1M Tris-Cl, 320ml water]
Pour the mess carefully into clean centrifuge tubes.
Put on ice and watch for at least 10 min for the solution to become viscous.
Take the cap off and see the snotty DNA.
Balance the tubes to within 0.01g.

Spin the tubes at 30,000rpm for 30 min at 4oC.
Make sure the rotor is clean and dry, and that the O-ring is seated properly.

Decant the supernatant into a 15ml disposable tube. Don't let pellet fall.
Estimate the volume by the gradations on the tube to the nearest 0.1ml.
Add 0.95g CsCl per ml. Invert gently until completely dissolved.

Into disposable ultracentrifuge tubes, pipet ethidium bromide. Wear gloves.
Approximately 200ul of 10mg/ml ethidium bromide per 10ml of CsCl/DNA solution.

Pipet the CsCl/DNA solution into the tubes. Use a pasteur pipet as a funnel.
Bring the volume up to the neck with a separate solution of CsCl.
This is made by adding 0.95g CsCl per ml to 25% sucrose, 50mM Tris.

Balance the tubes to within 0.01g. Seal the tubes.

Put the tubes in a clean,dry rotor.

If necessary, cover the tubes with metal caps that weigh the same.
Make sure the O-ring is seated properly.

Spin 50,000rpm at 15oC for about 36 hours.

Break down to 1000-800rpm, then let brake off.

Remove tubes carefully. Wear gloves.
Gently open the tubes at the top.
Insert a 21-20G needle in 3ml syringe just below lower band.
Pull lower band, remove needle carefully, put solution into polyallomer 5ml tube.
Empty tube into bleach. Discard needle in sharps bucket.

Extract solution at least twice with isopropanol over salt-saturated water.
Dialyze or precipitate the DNA. To precipitate, double the volume with TE;
add 2 volumes ethanol; ice, spin.

Large Scale Plasmid Midi Prep DNA Fingerprinting

Abstract:

The isolation of large amounts of plasmid DNA from insert-containing clones is necessary because subsequent DNA sequence analysis requires high levels of pure starting DNA. We will be using a moderately expensive plasmid isolation kit (Promega, Inc.) because it produces DNA compatible with our sequencing apparatus.  As compared with the rapid mini-prep, large-scale plasmid isolation methods all share three basic parts: (1) a way to gently lyse the cellular hosts; (2) a way to crudely separate plasmid from the total cell extract; and (3) a way to purify and concentrate plasmid DNA.

In order to compare your samples using DNA fingerprinting methods - you will set up restriction digests based on some moderately tricky pre-lab analysis. Recall that restriction enzyme digest reactions require template DNA, enzyme(s), an appropriate enzyme buffer, and some water. Today, you will set up uncut samples, single digests, and double digests using interesting enzymes that should reveal more complex cutting patterns than those produced by EcoRI last time. As you will see, there are many challenges to this. For example, two enzymes we want to cut at the same time may function to lesser extents in non-ideal buffers.  Charts are available in the form of company literature (in our case: Gibco BRL Life Technologies) to assist us when faced with the problem of making recipes and figuring these kinds of compatibility issues out. Additionally, you will analyze the enzyme's recognition sequence and calculate - based on probability - how many times each enzyme is predicted to cut the clone. Does the pattern you obtain match this prediction - why or why not?

See full Plasmid Isolation Procedures at

National Science Foundation

Western Oregon University

Yellowstone National Park

http://www.wou.edu/~boomers/Molecular/midi.htm

Isolation of Acetylcholinesterase and lectin Protocols Links*

Identificationandisolationoflectinnu.pdf
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IsolationandCharacterizationofRanaca.pdf
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Isolationandpartialcharacterizationo.pdf
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Isolationandpropertiesofalectinfromt.pdf
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Isolationofalectinfromthepericarpofp.pdf
521k

proteinpurificationmicrotomacro.pdf
1M

PurificationandCharacterizationofanN.pdf

 

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How to do Mixed-cell-culture assays for analyzing neuronal synapse formation

The assembly of synapses in the vertebrate central nervous system requires bidirectional signaling across the synaptic cleft that directs the differentiation of pre- and postsynaptic membrane domains. Biochemical and genetic studies have identified several adhesion and signaling molecules that localize to synapses and might participate in organizing synaptic structures. Understanding how individual proteins contribute to synaptic organization is complicated by the fact that there are significant numbers of separate signals that cooperate in this process. This protocol describes an assay system that permits examination of synaptogenic activities of individual cell-surface proteins in isolation. Besides the time needed for preparation and growth of primary neuronal cultures (6-14 days), the execution and analysis of the assay is rapid, requiring approximately 2 days. Using this assay, recent studies revealed that single synaptic adhesion complexes can direct a remarkable degree of synaptic differentiation and provided new insights into the cell biological mechanisms of synaptogenesis.

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