ISOLATION OF ENDOPLASMIC

RETICULUMPresenter:

Maria Khadija(BBI 151002)

ENDOPLASMIC RETICULUM

ENDOPLASMIC RETICULMSite where cell membrane and exported

material is made Netlike arrangement of flattened, hollow tubules

continuous with nuclear envelope Functions as transport system

TYPES OF ENDOPLASMIC RETICULM Rough endoplasmic reticulum(RER) smooth endoplasmic reticulum(SER)

FUNCTION 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 

PRECAUTIONS 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.

PROCEDURE 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.

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.

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

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.

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).

IMAGE SHOW ENDOPLASMIC RETICULUM

CON… 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.

CON.. 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.

RESULT Endoplasmic reticulum is separated…..

REFERENCES:  

www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/er0100bul.Par.0001.File.tmp/er0100bul.pdf

 http://biology.about.com/od/cellanatomy/ss/endoplasmic-reticulum.htm

micro.magnet.fsu.edu/cells/endoplasmicreticulum/endoplasmicreticulum.html

"The plant  Entoplasmic Reticulam ";  D.G .Robinson; springer;2006

"Plant Cell Biology : from Astronomy to zoology";  Randy Wayne; Elservier, inc; 2009

 

Any question??

Thank you