(a) While in the ER, only N-linked glycosylation can occur, which attaches carbohydrates to asparagine amino acids. There are 25 asparagine amino acids, so 25 carbohydrates will be attached in the ER. (b) While in the Golgi apparatus, both N-linked and O-linked glycosylation can occur. N-linked glycosylation attaches 25 carbohydrates (same as in ER). O-linked glycosylation attaches carbohydrates to serine and threonine amino acids, of which there are 25 each, for a total of 50. Therefore, the total number of carbohydrates attached in the Golgi apparatus is 25 (N-linked)

1. Introduction to Cell Biology
Part 4

2. Technique: Mass Spectrometry
1. The sample enters the mass spectrometer.
2. Vacuum inside the mass spectrometer prevents materials
from the air from reacting.
3. The ion source of the mass spectrometer bombards the
sample with electrons.
4. The electrons will cause some of the covalent bonds in the
sample to break, while others remain intact.
5. The mass analyzer of the mass spectrometer separates the
fragments according to size.
6. The detector of the mass spectrometer detects the
abundance of the fragments.
7. The computer software analyzes the fragments, constructs a
mass spectrum, and compares it with a library of
compounds of known mass spectra.
8. The mass spectrum is a graph of the abundance of the
fragment ions (y-axis) vs. the mass to charge ratio (x-axis).

3. Technique: Western blotting
1. After transferring the gel onto the membrane, the membrane is
blocked to prevent antibodies from sticking to locations where
the protein of interest is not found.
2. The membrane is treated with a primary antibody to locate the
protein of interest.
3. The membrane is washed to remove any excess primary
antibody.
4. The membrane is treated with a secondary antibody attached to
an enzyme horseradish peroxidase that will detect the primary
antibody.
5. The membrane is washed to remove any excess secondary
antibody.
6. The membrane is treated with an enhanced chemiluminescence
(ECL) reagent that reacts with the horseradish peroxidase.
7. The reaction is then detected by an X-ray film in the dark room,
and the X-ray film is developed to reveal the protein bands.

4. Technique: Electrophoretic Mobility
Shift Assay (EMSA)
• This technique is used to determine the following:
– Which proteins will bind to an approximate specific DNA
sequence (Protocol A)
– Which approximate DNA sequences will bind to a specific
protein (Protocol B)
• Protocol A
1. Several tubes filled with DNA of the same sequence are labeled
with 32P. The tubes are paired with each other.
2. One tube (of each pair) is incubated with a different protein,
while the other is left as it is.
3. Both tubes of all pairs are run in a polyacrylamide gel to
determine the proteins binding to the specific DNA sequence.
4. The radioactive DNA is detected by an X-ray film in the darkroom,
and the X-ray film is developed to reveal the DNA bands.
5. Any shift in the DNA band means that the protein is binding to it.

5. Technique: Electrophoretic Mobility
Shift Assay (EMSA)
• Protocol B
1. Several pairs of tubes filled with DNA of different
sequences are labeled with 32P. Each pair has the same
DNA sequence.
2. One tube (of the pair) is incubated with protein of
interest, while the other is left as it is.
3. Both tubes of all pairs are run in a polyacrylamide gel
to determine the approximate DNA sequence the
protein is binding to.
4. The radioactive DNA is detected by an X-ray film in the
darkroom, and the X-ray film is developed to reveal
the DNA bands.
5. Any shift in the DNA band means that the protein is
binding to it.

6. Question
• Protein A binds to DNA sequence A, while
protein B binds to DNA sequence B. Draw an
EMSA gel that will show these results.

7. Technique: Chromatin
Immunoprecipitation (ChIP)
• This technique is used to determine the approximate
DNA sequence a protein will bind to.
1. Incubate cells in formaldehyde to cross-link the DNA
and the protein of interest.
2. Add glycine to stop the cross-linking.
3. Remove the plasma membrane of the cells by adding
cell lysis buffer.
4. Remove the nuclear membrane of the cells by adding
nuclear lysis buffer.
5. Break DNA into smaller fragments in a process called
sonication.
6. The sample is blocked to prevent the primary antibody
from sticking to other things in the solution.

8. Technique: Chromatin
Immunoprecipitation (ChIP)
7. The sample is treated in primary antibody to locate the
protein of interest.
8. The sample is washed off to remove excess primary
antibody.
9. The sample is treated with secondary antibody
attached to beads to detect the primary antibody.
10. The sample is washed off with high salt buffers to
remove the secondary antibody from the beads.
11. Reverse cross-linking by adding RNase and NaCl.
12. Add proteinase K to degrade the protein samples.
13. Get the sequence of the DNA by running through a
machine known as sequencer.

9. Technique: DNA Footprinting
• This technique is used to determine the exact DNA sequence a
protein will bind to.
1. Two tubes filled with DNA of the same sequence (about 400bp
long) are labeled with 32P.
2. One tube is incubated with protein of interest, while the other is
left as it is.
3. Both tubes are treated with DNase I, which will cut DNA at any
location that is not bound to protein.
4. Both tubes are treated with stop solution, which will stop DNase I
from cutting.
5. Both tubes are treated with Proteinase K, which will degrade all
the proteins in the sample.
6. Long DNA from both tubes is precipitated by ethanol and
removed from short free nucleic acids generated by the cutting.
7. The DNA from both tubes is run in a urea gel to determine the
exact DNA sequence the protein is binding to.
8. The radioactive DNA is detected by an X-ray film in the darkroom,
and the X-ray film is developed to reveal the DNA bands.

10. Question
• The particular DNA sequence used for DNA
footprinting is 5’-ATCGGCTAATCG-3’. If the
protein binds to the sequence 5’-GGCTAA-3’,
draw the resulting gel after the experiment is
performed.

11. Parts of the Cell
10. Endoplasmic reticulum (ER)
– Network of flattened sacs and tubes called cisternae that
synthesize membranes
– Lumen or cisternal space is the space inside the ER.
– Rough ER contain ribosomes and embed proteins into a
membrane.
• Helps the protein fold properly
• Adds carbohydrates in a process called glycosylation
– Transitional ER sends proteins and lipids to the Golgi apparatus for
further processing.
– Smooth ER do not contain ribosomes and synthesize lipids.
• Contains enzymes that detoxifies drugs in liver cells
• Sarcoplasmic reticulum removes Ca2+ from the cytoplasm to
help muscle cells contract and relax.
– Transmembrane proteins become embedded in a membrane and
are eventually sent to the plasma membrane.
– Water-soluble proteins goes to the lumen of the ER and are
eventually sent outside the cell.

12. Endoplasmic Reticulum (ER)
• ER signal sequence in the protein gets recognized by a signal
recognition particle (SRP).
• SRP directs the protein and the ribosome to go to the ER.
• ER signal sequence gets cleaved off by a signal peptidase once the
protein enters the ER.
• The protein passes through an aqueous pore into the lumen of the
ER.
• The start transfer signal tells the ER to let the –COOH part of the
protein into the lumen.
• The ER signal sequence may act as a start transfer signal.
• If the ER signal sequence is internal, it does not get cleaved off.
• The stop transfer signal tells the ER to embed it into the
membrane and to let the –COOH part out of the lumen.
• A multipass transmembrane protein passes through the
membrane several times and has several start and stop transfer
signals.
• If a protein folds improperly in the ER, it goes to the proteasome
to be degraded.

13. Question
• The sequence of a protein is NH2-start-stop-
start-stop-start-stop-COOH. Draw the protein,
specifying the cytoplasm and the lumen of the
ER.

14. Parts of the Cell
11. Golgi apparatus
– Organelle involved in the synthesis, modification, sorting, and
secretion of cellular products
– Receives membranes with proteins from the ER and directs where
they go
– Site of carbohydrate synthesis; also involved in glycosylation
– Consists of cisternae divided into the cis compartment (entry
face), the medial compartment (middle), and the trans
compartment (exit face). Each stack is a dictyosome.
– Only proteins that are properly folded leave the ER to go to the
Golgi apparatus.
– In the vesicular transport model, the proteins move to the specific
compartment through transfer vesicles from the ER.
– In the cisternal maturation model, the actual cisternae bud off and
move from one compartment to the other.
– N-linked glycosylation attaches carbohydrates to asparagine
amino acids and occurs in the ER.
– O-linked glycosylation attaches carbohydrates to serine and
threonine amino acids and occurs in the Golgi apparatus.

15. Question
• A protein has 25 asparagine amino acids, 25
serine amino acids, and 25 threonine amino
acids. Determine how many carbohydrates are
attached to the protein (a) while it is in the ER
(b) while it is in the Golgi apparatus.