SDS-PAGE/Western immunoblotting:
Inducible nitric oxide synthase (iNOS)
Laboratory 3:
Thursday February 3, 2011
I. Introduction
A. SDS-PAGE:
SDS (sodium dodecyl sulfate) is a detergent with a net negative charge. When a
protein and SDS are mixed together, the hydrophobic tail of the SDS molecule
inserts into the hydrophobic region (center) of the protein and the negatively charged
portion of the molecule interacts with the protein surface. Thus the protein becomes
negatively charged due to its interaction with SDS regardless of the number of
charged R groups in the amino acids that make up the protein. SDS also destroys
tertiary and quaternary structure of a protein. Therefore, we can use SDS-PAGE
(polyacrylamide gel electrophoresis) to separate proteins based on differences in
size. Smaller molecules move through the polyacrylamide gel faster than larger
ones. Using proteins of known molecular weights (standards), we can determine the
relative molecular weights of the proteins present in cell lysates. Proteins can then
be transferred to a nitrocellulose membrane, and specific antibodies can be used to
detect proteins of interest.
B. iNOS:
The enzyme inducible nitric oxide synthase (iNOS) is responsible for producing
nitric oxide (NO) in macrophages as part of the inflammatory immune response in
order to destroy invading pathogens. When LPS binds to receptors (CD14/TLR4)
on the surface of a macrophage a signal transduction pathway is initiated and iNOS
is transcribed and translated into protein (along with many other genes/proteins
involved in the inflammatory response).
The signal transduction pathway involves the transcription factor, NFκB. Prior to
receptor activation NFκB,is bound by IκB in the cytoplasm (thus preventing it from
moving into the nucleus and initiating transcription). Once the pathway is turned on
IκB kinase (IKK) phosphorylates IκB which can then no longer bind NFκB.
Phosphorylated IκB is ubiquinated which tags it for destruction by the proteosome.
NFκB then is free to move into the nucleus and turn on genes that are involved in
the inflammatory response, such as iNOS. The level of iNOS that is found in the cell
reflects the ability of that cell to produce NO.
II. Purpose:
The purpose of this laboratory is to detect iNOS in cell lysates prepared from
activated macrophages.
1. Use SDS-PAGE to separate proteins found in the cell lysates.
2. Transfer separated proteins from the gel to a ntirocellulose membrane in order to
perform western immunoblotting analysis and identify a specific protein (iNOS)
found in the cell lysates.
3. Blots will also be stripped and reprobed for actin, a house-keeping protein, to
demonstrate equal protein loading in each lane of the gel.
A. Macrophage cell lysates: prepared last week
B. Supplies and reagents needed for SDS-PAGE:
The class will run 10 gels
9 gels will be transferred, 1 will be stained
1. mini-gel unit and accessories
2. power supply
3. 10% acrylamide gels
4. heating block
5. pipets and pipet aid
6. P20 and P200; tips
7. gel loading tips
8. tank buffer; 300 ml
9. 2X sample buffer
10. protein molecular weight standards
11. coomassie Blue stain
C. Supplies and reagents needed for electrophoretic transfer:
1. mini transblot unit and accessories
2. power supply
3. plastic dishes
4. filter paper
5. nitrocellulose
6. gels
7. transfer buffer, 1 liter
D. Protocol:
1. Assemble the mini-PROTEAN II cell unit to hold 2 gels.
2. Prepare samples (table below) and protein standards (5 μl, no sample buffer
needed) by adding appropriate volume of sample, dH2O and 2X sample
buffer to labeled microfuge tubes.
Sample conc. ? μl = 30 μg
lysate
1
2
3
4
5
6
dH2O + lysate 2X sample
= 25 μl
buffer (25 μl)
25 μl
25 μl
25 μl
25 μl
25 μl
25 μl
Total volume
= 50 μl
50 μl
50 μl
50 μl
50 μl
50 μl
50 μl
3. Heat samples and molecular weight markers in a heat block for 2-5 minutes at
95C.
4. Set up gel unit and add approximately 300 ml of tank buffer to the gel unit.
5. Load gels with samples and one lane of molecular weight markers using gelloading tips.
6. Run gels at 200 volts for 40 minutes or until the dye runs off the gels.
7. Remove gels. Place one in a dish containing Coomassie Blue stain (cover; we
will destain the gel next week) and the others in a dish containing transfer buffer
for 15 minutes.
8. Soak fiber pads, filter paper and nitrocellulose in a dish.
9. Assemble gel holder cassettes in the following manner:
a. place the dark side of the cassette flat in a dish
b. place a soaked sponge on the cassette
c. place a wet piece of filter paper on the sponge
d. place the gel on the filter paper; the left nicked corner should be on the right
(essentially, flip the gel)
e. place the wet nitrocellulose on the gel; smooth the membrane carefully; there
can be no air bubbles between the gel and the membrane
f. place the other soaked sponge on the membrane
g. close the cassette and insert into the unit; dark side of the cassette should be
against or close to the black side of the unit
h. insert the unit into the gel box
10. Add the cooling block and fill the box with transfer buffer.
11. Run the transfer for 1.5 hours at 100 volts or overnight at 30 V.
12. After the transfer is finished, remove membranes and allow them to air dry.
13. Label membranes (very carefully) and store in a safe, dry place until next week.
Helpful hint:
After running your gels nick the upper left corner of the gels in order to orient them
correctly for transferring to the membranes. After the transfer is complete nick the
membranes in a similar manner.
Next week:
Perform western immunoblotting analysis using an anti-iNOS antibody.
(Afterwards, we will strip the blots and probe them using an anti-actin antibody.)
Destain the gel.