Biochemistry _ serum protein separation

Biochemistry and molecular biology lab
Lecture 18
Serum protein separation by
cellulose acetate membrane
electrophoresis

Aim
 Learn the principle of cellulose acetate
membrane electrophoresis
 Know the operation and clinical significance of
electrophoresis

Principle
 Electrophoresis:
is the motion of charged particles relative to a
fluid under the influence of an electric field.
Factors determining the electrophoresis motion ：
charge, size, sharp
Based on different supporting materials, there are membrane
electrophoresis and gel electrophoresis
• Gel electrophoresis is the process by which molecules
in a sample can be separated by charge and/or size.

Lab equipments
 electrophoresis device
electrophoresis chamber DYY-2 model power supply
The connection between electrophoresis chamber and power supply

 cellulose acetate membrane
（2 cm×8 cm ）
 Petri dish：staining and rinsing
 Sample applicator (plastic slice)
 Filter paper
 Watch glass
 Forceps

 Rocking table
 722 model
spectrophotometer

Reagents
 fresh serum
 Barbitone sodium Buffer （pH8.6）
 Staining solution（amido black 10B）
 Destaining solution
 0.4mol/L NaOH

Experimental steps
1. Membrane
preparation
Soaking the membrane with
buffer for more than 30
mTinak；e the membrane with forceps, place the
membrane between two pieces of filter paper to
dry the buffer (not too dry), and differentiate
between the smooth and rough surface (rough
surface faces up).
Note：Only touch the margins of the
membrane

2. Sample spotting：
Take a small amount of serum with the plastic slice, and stamp on the membrane
Notes：
Mark with
pencil
 Spotting on the rough surface, 1.5cm from one membrane short
edge
 softly press for 1-2 sec, let the serum absorbed by the membrane
 Spotting only once, no need to repeat
 Spot requirements: thin, straight and not reaching the long edge

3. Place the sample：
Connect the power supply with electrophoresis chamber
Let the rough surface faces down, the spotting side is
placed at the cathode (-) side (Note: do not let the spot
overlap with the supporting paper)
Remove bubbles between the membrane and supporting
paper

4. Electrophoresis ：
 Make sure that the membrane is wet
 Pre-electrophoresis：50V, 5min
 Electrophoresis：Stable voltage, 110V, 40 min

4. Staining and rinsing ：
Staining：Transfer the membrane using forceps to the Amido black 10B
and stain for 3 min
Note：Only touch the margin regions of the membrane with
forceps；Completely submerge every membranes into the staining
solution, no overlapping.
Rinsing：detaining on the rocking table for 3 times
(8 min; 7 min; 6 min)
serum albumin globulins Starting point

5. Quantification：
Cut each bands and a part of the blank membrane, add 4ml
NaOH respectively and shake for 15 min. Determine the light
absorbance at 620 nm.
6. Calculation：
Total absorbance： T= A +α1+ α2+ β+γ
Percentage = ( X/ T ) ×100%
A/G = A/(α1+ α2+ β+γ)

Results
serum albumin
α1 -globulins
α2 -globulins
β- globulins
γ- globulins

Normal range
• serum albumin 57.45-71.73%
• α1-globulins 1.76-4.48%
• α2-globulins 4.04-8.28%
• β-globulins 6.79-11.39%
• γ-globulins 11.85-22.97%
• A/G 1.24-2.36

Clinical significance
 Most of serum protein is produced by liver ，only
γ-globulins produced by plasma cells
 The function of serum proteins ：
Maintain plasma colloid osmotic pressures; maintain
plasm pH balance, base-acid balance; Transport
nutrients, metabolites, hormones, medicines and metal
ions.

 liver cirrhosis：serum albumin, α1, α2 ↓，γ- globulins
↑↑；
 Hepatocarcinoma：between albumin and globulins,
there is an alpha feto protein (AFP) band ；
 acute and chronic nephritis & nephrotic syndrome ：
serum albumin ↓ ， α2 and β globulins ↑；
 Multiple myeloma ： serum albumin ↓ ，γ- globulins
↑ ，between β and γ- globulins, there is a “M” band

Assignment questions
1.Which side (anode or cathode) shall we place
the sample during electrophoresis? Why?
2. What are the possible reasons causing the
irregular, distorted or atypical electrophoresis
bands？

Other electrophoresis techniques ：
1、SDS-Polyacrylamide Gel Electrophoresis
usually used for protein molecular weight
determination

2、Isoelectric focusing electrophoresis：
a technique for separating different molecules by differences in
their isoelectric point (pI)
3、2D electrophoresis：
an important technique in the proteomics
4、Agarose gel electrophoresis：
DNA or RNA separation

Agarose Gel Electrophoresis
Gel electrophoresis is a widely used technique for the
analysis of nucleic acids and proteins. Agarose gel
electrophoresis is routinely used for the preparation and
analysis of DNA.
Gel electrophoresis is a procedure that separates molecules on the basis of
their rate of movement through a gel under the influence of an electrical field.
We will be using agarose gel electrophoresis to determine
the
presence and size of PCR products.

Biochemistry and molecular • DNA is negatively charged. biology lab
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by
size.
- +
Power
DNA
• Polymerized agarose is porous,
allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (1×1 μm) 

How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
- +
Power
DNA
small
large
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.

Biochemistry and molecular Agar boioslogey lab
D-galactose 3,6-anhydro
L-galactose
•Sweetened agarose gels have
been eaten in the Far East since
the 17th century.
•Agarose was first used in biology
when Robert Koch used it as a
culture medium for Tuberculosis
bacteria in 1882
Agarose is a linear polymer extracted from seaweed.

Making an Agarose Gel
An agarose gel is prepared
by combining agarose
powder and a buffer
solution.
Buffer
Agarose
Flask for boiling

Casting tray
Gel combs
Power supply
Gel tank Cover
Electrical leads

Electrophoresis Equipment

Gel casting tray combs

Preparing the Casting Tray
Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.

Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.

Melting the Agarose
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and may
boil violently if it has been heated too long in a microwave oven.

Pouring the gel
Allow the agarose solution to cool slightly (~60ºC) and then
carefully pour the melted agarose solution into the casting
tray. Avoid air bubbles.

Each of the gel combs should be submerged in the melted agarose solution.

When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.

Place the gel in the electrophoresis chamber.

buffer 

wells
Cathode
(negative)
Anode
(positive)
DNA
  
Add enough electrophoresis buffer to cover the gel to a depth of
at least 1 mm. Make sure each well is filled with buffer.

Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking
dye). This allows the samples to be seen when loading onto the gel, and
increases the density of the samples, causing them to sink into the gel
wells.
6X Loading Buffer: 
· Bromophenol Blue (for color)
· Glycerol (for weight)

Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.

Running the Gel
Place the cover on the electrophoresis chamber, connecting the electrical
leads. Connect the electrical leads to the power supply. Be sure the leads
are attached correctly - DNA migrates toward the anode (red). When the
power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.

Cathode
(-)
 wells
 Bromophenol Blue
DNA
(-)

Anode
(+)
Gel
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.

DNA Ladder Standard
 12,000 bp
 5,000
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100
-
Note: bromophenol
blue migrates at
approximately the
same rate as a 300 bp
DNA molecule
bromophenol blue
+
DNA
migration
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy
to determine the sizes of unknown DNAs.

Biochemistry and molecular biology lab Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.
***CAUTION! Ethidium bromide is a powerful mutagen and is
moderately toxic. Gloves should be worn at all times.

Safer alternatives to Ethidium Bromide
· Methylene Blue
· BioRAD - Bio-Safe DNA Stain
· Ward’s - QUIKView DNA Stain
· Carolina BLU Stain
…others
advantages
Inexpensive
Less toxic
No UV light required
No hazardous waste disposal
disadvantages
Less sensitive
More DNA needed on gel
Longer staining/destaining time

Staining the Gel
• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.

Ethidium Bromide requires an ultraviolet light source to visualize

Visualizing
the DNA
(ethidium
bromide)
DNA ladder
 5,000 bp
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100

DNA ladder

PCR Product
1 2 3 4 5 6 7 8
wells
+ - - + - + + -
Primer dimers
Samples # 1, 4, 6 7 were positive

Visualizing the DNA (QuikVIEW stain)
DNA ladder
 2,000 bp
 1,500
 1,000
 750
 500
 250

wells
PCR
Product
+ - - - - + + - - + - +
Samples # 1, 6, 7, 10 12 were positive

Biochemistry _ serum protein separation

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