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Electrophoresis Instrumentation

Overview of components for CLS 447
by

Jessica Wireman

on 13 March 2014

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Transcript of Electrophoresis Instrumentation

Overview
Instrument Components
Electrical Power
http://wealteceurope.com/electrophoresis.html
http://commons.wikimedia.org/wiki/File:Electrophoresis_power_supply_(2).JPG
http://commons.wikimedia.org/wiki/File:Electrophoresis_power_supply_(5).JPG
Support Medium
Buffers
Procedure
Testing concepts
References
Electrophoresis Instrumentation
Heather Brown
Jessica Wireman
Study Guide
Concepts, Terms, & Models
Instrument Components
Samples & Procedure
Testing Concepts & Board ?s
wikipedia.org/wiki/File:Two_percent_Agarose_Gel_in_Borate_Buffer_cast_in_a_Gel_Tray_(Front,_angled).jpg
https://proteabio.com/products/BR-000
Agarose
Polyacrylamide
https://www.nzytech.com/site/vmchk/Buffers/TAE-Buffer-50x-pH-83
TAE (Tris-acetate-EDTA)
ClearPAGE TGS Transfer Buffer
http://www.cbsscientific.com/fb82500clearpagetgstransferbufferbuffer.aspx
Cellulose
Gather supplies
Check installation
Set up and map creation
Apply sample
Electrophoresis
Dry
Stain
Quantify
Analysis
Sample Troubleshooting Issues
Sample application
http://www.slideshare.net/rachellraines/electrophoresis
What is Electrophoresis?
A principle not a machine
Current
Resistance
Voltage
Electroendosmosis
Amphoteric
Cations, H+, Acid, Cathode(-)
Anions, OH-, Bases, Anode(+)
Isoelectric Charge
TERMS
&
Concepts
Power Issues
Medium Issues
Troubleshooting pH
Electroendosmosis
Choice
Stability
Insufficent Blotting
Opens
Shorts to ground
High resistance
pH, Binding or Losing H+
Electroendosmosis
Scanner Densitometer
&
Interpretation
Scanner Issues
sample sources
urine
blood
plasma
CSF
serum
buccal cells
Board Questions
#216 MLS Only pg. 458

On electrophoresis, distorted zones of protein separation are usually due to:

a. Presence of therapeutic drugs in serum sample
b. Dirty applicators
c. Overloading of serum sample
d. Prestaining with tracer dye

#313 pg. 122
When separating serum proteins by cellulose acetate electrophoresis, using Veronal™ buffer
at pH 8.6, beta globulin migrates:

a. Faster than albumin
b. Slower than gamma globulin
c. Faster than gamma globulin
d. Faster than alpha-2 globulin

#314 pg. 122

Hemoglobin S can be separated from hemoglobin D by which of the following methods?

a. Agar gel electrophoresis at pH 5.9
b. Thin-layer chromatography
c. Alkali denaturation
d. Ammonium precipitation

#316 pg. 122

The buffer pH most effective at allowing amphoteric proteins to migrate toward the cathode in an electrophoretic system would be:

a. 4.5
b. 7.5
c. 8.6
d. 9.5

#317 MLS Only pg. 122

On electrophoresis, transient bisalbuminemia or a grossly widened albumin zone is associated with:

a. Dirty applicators
b. Presence of therapeutic drugs in serum sample
c. Endosmosis
d. Prestaining with tracer dye

#318 MLS Only pg. 122

Which of the following serum proteins migrate with the beta-globulins on cellulose acetate at pH 8.6?

a. Ceruloplasmin
b. Hemoglobin
c. Haptoglobin
d. C3 component of complement

The middle sample in this electrophoretic strip has adark elliptical spot overlying the a2- to b1-region (arrow). Thisrepresents a drop of serum falling on the gel (about in thea1-region) prior to electrophoresis.
High-resolution electrophoretic strip with four samples that all appear to have a small, slow g-restriction (arrow in restriction of top lane only). If such a band were present in only one sample, a monoclonal gammopathy should be suspected. However, when it occurs in more than one, it represents an artifact. In this case, the artifact results from excess buffer in the reservoir
Excessive Buffer Case Study
Skewed or distorted bands
: this can result by an uneven heating of the gel or even issues with the gel itself.
Poor resolution of bands (fuzziness)
: current ran too high or too large of a sample used.
If bands bend in a smiley face pattern:
This can indicate too high of voltage used.

This is an example of a normal patient band pattern and how the peaks would appear on a densitometer.
http://www.medialabinc.net/spg422884/migration_of_hemoglobin_in_acid_electrophoresis.aspx
http://www.medialabinc.net/spg422879/migration_of_hemoglobin_in_alkaline_electrophoresi.aspx
The sample shows two faintly stained bands in the a1-region (arrows) compared to the normal a1-region in the other serum.
A densitometric scan indicates that the a1-region abnormality observed to the left can be seen (arrows) by enhancing the sensitivity of the densitometer.

The third sample from the top is a case of bisalbuminemia. Note that the two bands did not separate completely on this gel. The fact that there are two bands present can be noted by the indentation (arrow) between the two bands. (Paragon SPE2 system stained with Paragon Violet.)

http://www.bio.davidson.edu/COurses/genomics/method/Capillary.html
http://www.bio-rad.com/en-us/applications-technologies/first-dimension-separation-isoelectric-focusing
C is the correct answer:
Act as a carrier for ions

Electrophoresis involves the migration of a charged molecule or particle in a liquid medium under the influence of an electric field.
The 3 roles of the buffer are
1) to carry the applied current
2) to establish the pH for the electrophoresis procedure
3) to determine the electrical charge on the solute.
C is the correct answer;
Overloading of the Sample

A commonly encountered problem in electrophoresis is holes seen in staining patterns. This is due to the analyte being present in too high a concentration. The corrective action is to apply a less concentrated sample.
B is the correct answer;
occasionally, a split albumin zone is observed in the rare benign genetically relation condition of bisalbuminemia. However, transient bisalbuminemia or a grossly widened albumin zone could be due to albumin-bound medications.
A is the correct answer

Proteins are amphoteric substances; that is, they contain acidic and basic groups. Their overall (net) charge is highly positive at low pH values, 0 at particular higher pH, and negative at still more alkaline pH values. At a pH of 4.5, the positively charged proteins will migrate toward the cathode in an electrophoretic system.
A is the correct answer and acidic agar shows the best Hgb S seperation from Hgb D.
C is the correct answer; protein electrophoresis migration.
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/smear.html
Acetate
Cellulose
SLOWEST
FASTEST
Isoelectric Point
- http://www.pathology.ecu.edu/Public/faculty/epbookpict.pdf
-http://lclane.net/text/geltips.html
-http://www.jpma.org.pk/full_article_text.php?article_id=2591
-http://www.merriam-webster.com/dictionary/phoresis?show=0&t=1393989125
-http://o.quizlet.com/i/QA5dT-2MuLbPk24GW1DooA_m.jpg
- BOC Study Guide Clinical Lab Certification Exams
-Clinical Chemistry Techniques Principles, Correlations 6th edition. Bishop 
-Keren, F. David. Protein Electrophoresis in Clinical Diagnosis. -Hodder Arnold 2003, pgs. 69, 31, 114, 117, 119.
http://www.slideshare.net/biotechvictor1950/electrophoretic-techniques-for-life-science-researchers
-http://www.biochemia-medica.com/content/bisalbuminemia-male-croatian-patient-sarcoidosis
-http://www.emelysfilms.com/misc/.../Elect%20Sep%20-%20west%20blot.doc‎
-Capillary http://www.bio.davidson.edu/COurses/genomics/method/Capillary.html
-Isoelectric http://www.bio-rad.com/en-us/applications-technologies/first-dimension-separation-isoelectric-focusing
-http://www.thefreedictionary.com/amphoteric
-http://quizlet.com/5276377/serum-proteins-images-flash-cards/
#215 pg. 458 BOC Book

In electrophoretic analysis, buffers:

a. Stabilize electrolytes
b. Maintain basic pH
c. Act as a carrier for ions
d. Produce an effect on protein configuration
ROBERT
Christina
Jeremy
Cynthia
Patricia H
Jeremy
Robert
a. Faster than albumin
b. Slower than gamma globulin
c. Faster than gamma globulin
d. Faster than alpha-2 globulin
- Phoresis
To carry, Transmission
The migration of molecules through a support medium under the influence of an electric field
TYPES OF ELECTROPHORESIS
alpha1 anti- trypsin
alpha1 acid glycoprotein
alpha2 macroglobulin
Haptoglobin
Hemopexin
Transferrin
Beta Lipoprotein
Complement C'3
IgA
IgM
IgG
D is the correct answer;
the C3 component of complement migrates with beta-globulins on electrophoresis.
Om's Law

a. Ceruloplasmin
b. Hemoglobin
c. Haptoglobin
d. C3 component of complement
Name two test that you may run in the Lab using an electroporesis instrument
Cynthia
AMPHOTERIC
Having the characteristics of an acid and a base and capable of reacting chemically either as an acid or a base.
Albumin 4.8
Gamma Globulin 7.2
Albumin 4.8
Gamma globulin 7.2
pH 8.6
Full transcript