1. Lysate extraction

Modified RIPA

a) DLD1 colorectal cancer cells were grown on 100 mm plates until cells were 80% confluent. The medium was removed and the cells were scraped from the surface by gradually adding 500μl of the lysis buffer(modified RIPA).On prolonged exposure RIPA ruptures the membranes of the cell.

2. Protein Estimation

Absorbance

0.622

Standard curve

abso

rban

ce

concentrations

b)Protein was estimated by BCA method ,using the Pierce kit. This assay works on the principle of colorimetry and gives corresponding absorption values for known concentration of protein . The calibration curve thus obtained is used to estimate the concentration of any given protein sample.

C)The estimated protein was alliqoted into small vials ,each containing 500 μ g of protein lysate ,and 1.5 μg of Lamin B1 Rb α antibody (1 μg/ μl) was added to one such vial. The vials were incubated overnight at 4 8C on a rocker.

Lamin B1 antibody raised in rabbit

Y YYYY

YY

Whole cell lysate

500μg of protein lysate, as control.

500μg of protein lysate, as test.

alliquoted

incubation

3. ANTIBODY ADDITION

Y

Y

Y

Y

Agarose beads coated with Protein A

Incubated ,

for 3 hours

d) 100 μl of the Protein - A coated agarose beads which were in the form of a 50% slurry were washed in 1 x PBS and added to each of the vials. The vials containing the protein-antibody complex and the beads suspended in ~25 μl 1X PBS were incubated for 3 hours at 4 8C on a rocker.

4. CAPTURING THE IMMUNOCOMPLEX

4 a . Beads addition

Removal of the supernatant .

Addition of dye, followed by heating and centrifugation at 4 8C ,14000 rpm .

Y

Y Centrifugation

4 8C ,14000 rpm.

Y

Y

Supernatant

pellet

Y

Y

Y

Ysupernatant

Collect supernatant

Y

Y

4b. Collecting the complex attached to the beads

4b. The samples were centrifuged so that the immunocomplex attached to the beads settle down .To the pellet thus extracted was first washed with mod.RIPA to remove weak interactions ,if any and 60 μl of 2X loading dye was added and heated to dissociate the beads .Dissociated beads can be separated by pulse centrifugation.

5.Analyzing the constituents of the immuno-complex. A) SDS-PAGE

1]Coomassie staining ,to detect different proteins in the immuno complex

2] western blot to detect the presence of specific binding

3]Silver staining for a more sensitive detection of protein ,which can be sent for analysis by mass-spectroscopy.