Investigation of identity

Sumanpreet- general project plan

Investigation of identity of Cltx membrane binding partner in MDA MB-231 and U251-MG cancer cell lines

Investigate the effects of VEGF and ClTx on cancer cell proliferation

Investigate NRP1 as CXltx membrane-binding partner on cancer cell lines

General plan

Week Cell culture strand Western blotting and Cltx binding partner strand
1

Get cells growing

Practice counting cells

Trypan blue assay

Getting started in the lab sheet

BCA protein assay practice

SDS PAGE practice with FCS or BSA

2

Initial cell titre glo assay using 5000 cells/well in serum and in serum free media.

Make cell lysates

Calculate protein concentration

Read up about total protein staining for WB standardisation

3

May need to adjust cell number

Effect of VEGF in +/- serum conditions

WB for NRP1

Check expression in your cell lines

Work out how much lysate (and cells) need for later exps

4

Further tests on effects of VEGF

Diff concs

Effect of Anti-VEGF

Make Cltx conjugate

Cross linking exps and analysis

Internalisation

5

Cltx/VEGF/ anti VEGF treatment combos

6

Contingency/replicate week

7

Contingency/replicate week

Additional exps

Contingency/replicate week

Additional exps