The document describes the process of isolating endoplasmic reticulum from HeLa cells. It involves growing HeLa cells in a T150 flask, treating with trypsin/EDTA to detach the cells, centrifuging to pellet the cells, homogenizing the cells to lyse them, and then centrifuging multiple times at increasing speeds to separate out the crude microsomes containing the endoplasmic reticulum. Precautions like sterilizing equipment and proper labeling of materials are also outlined.

2. Presenter:
Maria Khadija
(BBI 151002)

4. Site where cell membrane and exported
material is made
 Netlike arrangement of flattened, hollow tubules
continuous with nuclear envelope
 Functions as transport system

5.  Rough endoplasmic reticulum(RER)
 smooth endoplasmic reticulum(SER)

6. SER include steroid metabolism, regulation
of calcium concentration, metabolism of
carbohydrates, synthesis of lipids and
steroids, drug detoxification
 detoxification of certain drugs and harmful
substances

7.  1. Before starting the experiment sterile the
laminar air flow chamber using spirit.
2. All the tubes and bottles used in the
experiment should be labeled properly.
3. Always disinfect your work area when you
are finished.

8.  Take out a T150 flask containing the adherent (HeLa)
cells (2x 108) from the carbon dioxide incubator.
 Remove the media from the T150 flask completely and
add 20 ml of 1x PBS.
 Remove the PBS and add 4ml trypsin/EDTA solution
to the flask and incubate for 2 minutes in a carbon
dioxide incubator.

9.  Observe the cells under the phase contrast
microscope to check the detachment of
cells.
 Add 16 ml cell culture media into the flask
and mix gently.
 Transfer the entire contents into a 50 ml
centrifuge tube and centrifuge at 1000 rpm
for 4 minutes.

10.  Remove the supernatant and wash the cell
pellet once with 20ml 1xPBS.
 Centrifuge again at 1000rpm for 4minutes
and add 1.2 ml of 1xPBS to the cell pellet
 Transfer the contents into a 1.5 ml microfuge
tube and centrifuge at 1000rpm for 4
minutes

11.  Discard the supernatant and keep the cell
pellet in ice.
 Homogenize the cells with 1.2 ml of pre
cooled 1x extraction buffer in a dounce
homogenizer .The dounce homogenizer
should be rinsed in pre cooled 1x extraction
buffer before homogenization.

12.  Give 10-20 strokes with the dounce
homogenizer.
 Check the cell lysis on a glass slide with
cover slip till 70-80 % cells are broken.
 Observe under the microscope to check
whether the cells are broken. Lysed cells
look pale in color (in a glass slide).

14.  Transfer the homogenized cells into a 1.5 ml
precooled microfuge tube and Centrifuge at
4degree C for 10 minutes at 2500 rpm
 Collect the supernatant in a fresh tube and
centrifuge at 14,000rpm for 15 minutes.

15.  the supernatant in a fresh 1.5 ml
ultracentrifuge tube and Centrifuge at 43000
rpm for 1 hr at 4 degree Celsius in an
ultracentrifuge.
 Discard the supernatant and store the cell
pellet in 1x extraction buffer in -20 degree.
Colorless pellet will contain the crude
microsomes.

17.  Endoplasmic reticulum is separated…..

18.  www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/e
r0100bul.Par.0001.File.tmp/er0100bul.pdf
 http://biology.about.com/od/cellanatomy/ss/endoplasmic-
reticulum.htm
 micro.magnet.fsu.edu/cells/endoplasmicreticulum/endoplas
micreticulum.html
 "The plant Entoplasmic Reticulam "; D.G .Robinson;
springer;2006
 "Plant Cell Biology : from Astronomy to zoology"; Randy
Wayne; Elservier, inc; 2009
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