Dr. Ash Stuart
Lab Protocols
Cell & Molecular Biology
SBIO 406
Experiment 4
In order to study the cells and its functions, it is often necessary to isolate and characterize biological
membranes. The plasma membrane of mammalian erythrocyte has been one of the most widely studied
membranes, mainly because of the availability of erythrocytes and the ease with which pure plasma
membrane preparations can be obtained from them. Erythrocyte is a highly specialized cell that has
few, if any, organelles, so very pure plasma membrane preparations can be obtained without
contamination by organellar membranes.
This experiment features a three-stage isolation of plasma membranes from bovine erythrocytes:
Stage 1:
The Red Blood Cells (RBCs) are separated from blood by a low-speed centrifugation and
subsequent washing to remove plasma.
Stage 2:
The washed RBCs are transferred from an isotonic buffer to a buffer of low osmolarity,
causing cell lysis.
Stage 3:
The lysed cells are washed extensively to remove hemoglobin and other cytoplasmic
The resulting particulate material obtained from this preparation consists almost exclusively of
erythrocyte membranes. A strong ionic detergent, SDS, is used to disrupt and dissolve the components
of the erythrocyte membranes. The resultant solubilized proteins can then be resolved by SDS PAGE
(Polyacrylamide Gel Electrophoresis). Under these conditions the proteins separate according to the
molecular weights of the individual polypeptide chains.
Dr. Ash Stuart
Lab Protocols
Cell & Molecular Biology
SBIO 406
Materials/ Reagents/ Supplies
Cold centrifuge capable of 1,000 to 20,000 x g spins set at 4oC
Oakridge tubes (autoclaved)
bovine / sheep blood
Container with appropriate reagent for blood disposal
Isotonic Phosphate Buffer (310 mM pH 6.8)
Hypotonic Phosphate Buffer (20 mM pH 6.8)
Heating block or water bath set to 100o
Protein Mol. Wt standards (Both, low and high range)
Pre-cast gels (7.5%) and vertical electrophoresis apparatus for PAGE
Syringe with a 23 gauge needle, bent at 90 degrees in the middle
Protein Dye buffer
Tris-Glycine Buffer (to be made fresh right before or during the lab)
Protein-gel Staining Solution
Fixative/ destainer
Autoclaved Eppendorf tubes
Micropipettors, Autoclaved Tips, Pipettors, 5ml and 10ml pipettes
50 ml polypropylene tubes
250 ml Beaker
50 ml Measuring cylinder
Light Box
Camera and Polaroid Film
The experiment is to be performed by a team of four students.
IMPORTANT: Unless specifically instructed, perform all the procedure at 0 - 40C and use Gloves.
A. Preparation of Erythrocyte Membranes (Day 1):
1. Add 5 ml of isotonic buffer to 20 ml of fresh blood. Mix gently and centrifuge the mixture at 1,000
x g (calculate appropriate rpm for your centrifuge) for 10 minutes at 40C in Oakridge tube.
2. Carefully aspirate the plasma and buffy coat from the pelleted RBCs
3. Wash the cells by gentle inversion in isotonic buffer (add up to 20 ml of buffer) followed by
centrifugation for 10 minutes at 1,000 x g at 40C.
4. Aspirate the supernatant carefully and then repeat step 3.
5. Aspirate the supernatant carefully.
6. Add 30 ml hypotonic buffer to the above pellet. Gently agitate to mix the cells. Do not shake.
7. Centrifuge the lysed cells for 30 minutes at 20,000 x g (appropriate rpm) at 40C
8. Decant the supernatant, save the pellet. (Careful, loose pellet !!)
9. Resuspend in 25 ml of hypotonic buffer, centrifuge for 20 minutes at 20,000 x g at 40C.
10. Repeat steps 8 & 9, TWICE.
11. Finally, resuspend the pellet in 1 ml isotonic buffer. Do not resuspend any red residue of unlysed
12. Determine the total amount of protein by BioRad reagent. (Your instructor might ask you to skip
this step. Generally, you will get ~10 - 20 mg of protein/ml)
Dr. Ash Stuart
Lab Protocols
Cell & Molecular Biology
SBIO 406
B. PAGE of Membrane Proteins (Day 2):
1. Dilute the membrane preparation to obtain the protein concentration to ~5 micrograms /ml
2. Pipette a range of 5 to 20 microliters (the ideal amount to be loaded is ~100 micrograms protein per
gel lane) of the diluted membrane preparation in autoclaved eppendorf tubes and add 5 to 20
microliters of 2x protein dye as appropriate.
3. Snap-close the tubes and boil at 1000C for 5 minutes.
4. Quickly spin down the contents and save the tube on ice
5. Prepare the 7.5% polyacrylamide gel and set it up the sandwich in the gel box as demonstrated by
the instructor.
6. Wash the wells and remove the bubbles with Tris-Glycine Buffer in a syringe.
7. Load the boiled samples (step 4) on to the gel as shown by the instructor.
8. Also load the molecular weight markers (10 microgram each protein)
9. Fill up the tank as per instructions with the Tris-Glycine buffer.
10. Connect the vertical electrophoresis apparatus to the power supply unit.
11. Turn on the Power supply unit. Apply 100 constant volts for 60-120 minutes. (Careful !!! High
Voltage !!!!)
12. Monitor the electrophoretic run by observing the movement of bromophenol blue dye. Stop when
the dye reaches about one centimeter from the lower edge
13. Turn off the power before proceeding further.
14. Disconnect the apparatus from Power unit, take out the gel sandwich. Place it under the running
cold tap water for two minutes.
15. Very carefully remove the plates from the sandwich as instructed, cut out and discard a small piece
of gel from the lower right corner.
16. Place the gel in 100 ml of the staining solution (Coomassie Blue in 0.05% in Fixative/Destainer) in
a plastic box-tray for 30 minutes to overnight.
17. Drain the staining solution and destain in 100 ml Fixative/Destainer. You will have to make several
changes of the Fixative/Destainer till protein bands are visible against clear background.
18. Put the gel on the light box and take a picture.
C. Analysis of the results:
1. Paste the gel picture in your lab note book, label all the lanes and known molecular weights.
2. Measure the migration distance of all the major bands as instructed and plot a graph between log10
of Molecular weight vs. Migration distance for the Molecular weight marker lane. This graph will
represent the standard curve for molecular weights on this gel.
3. Using this standard curve and calculate the Molecular weights of the major erythrocyte membrane
protein bands separated by PAGE. You could use Dr. Petro's tutorial (that is linked from your
CMB lab website) to help you with this step.
4. Compare your results with known protein composition of erythrocyte membranes and identify the
proteins that you have separated. Now since the actual molecular weights of the membrane proteins
are also given to you at the end of this handout, you could compare the values that you obtained
from step 3 with the actual values. You need to calculate percent error for each protein (and explain
the difference in the discussion). You can do this step by drawing an appropriate table as
explained in the class. A sample of the table is given at the end of this handout.
5. Explain any discrepancies or unresolved proteins.
6. Explain what you learnt from this exercise.
Dr. Ash Stuart
Lab Protocols
Cell & Molecular Biology
SBIO 406
Did you analyze intact membrane-proteins or individual polypeptides of the proteins with this method?
Given below is a sample of SDS-PAGE of RBC membrane proteins. It will be helpful in identifying
the bands that you will obtain after your experiment.
SDS polyacrylamide gel electrophoresis pattern of the proteins in the human red blood cell
membrane. The gel in (A) is stained with commasie blue. The positions of some of the major proteins
in the gel are indicated in the drawing in (B); glycophorin is shown in red to distinguish it from band 3
(anion channel protein). Other bands in the gel are omitted from the drawing. The large amount of
carbohydrate in glycophorin molecules slows their migration so that they run almost as slowly as the
much larger band 3 molecules.
Dr. Ash Stuart
Lab Protocols
Cell & Molecular Biology
SBIO 406
Here is a sample gel picture from a previous semester. It has been labeled to help you identify the major
erythrocyte membrane proteins. The first lane on the left is that of the standard molecular weight marker.
S. No.
Protein Name
Calculated Mol. Wt. Actual Mol. Wt. Percent Error