Immunohisto and western blotting

Question 1

difference between immunohistochemistry and western blotting

Answer 1

• immuno = involves using a thin slice of formalin stained tissued
• WB= involves using homogenise/ lygase

Question 2

direct immunohisto

Answer 2

1) primary antibody added against antigen of interest
2) primary antibody is conjugated with enzyme which produces a colour change when substrate added
3) if colour change occurs - antigen present

Question 3

positives of direct methods for both immuno and WB

Answer 3

• less steps- quicker
• useful when antigen is abundant
• more specific

Question 4

negatives of direct methods

Answer 4

• expensive- requires access to diff specific labelled antibodies
• label may interfere with Ab binding

Question 5

indirect immunohisto

Answer 5

1) primary antibody against antigen of interest
2) secondary antibody raised against Ig from A DIFFERENT ANIMAL binds to primary antibody
3) secondary antibody is conjugated with a reporting enzyme- when substrate added- colour change

Question 6

positives of indirect methods

Answer 6

• cheaper
• more sensitive
• signal amplification- useful when low abundance

Question 7

basic protocol for Immunohisto

Answer 7

1) block non specific sites using bovine serum albumin (BSA)
2) wash with phosphate buffer solution
3) add primary antibodies against antigen
4) wash with PBS
5) add secondary antibodies against antigen
6) wash with PBS
7) visualise signal

Question 8

ELISA is better with

Answer 8

quantification

Question 9

outline western blottign

Answer 9

1) lyse cells
2) separate using electrophoresis on SDS-page
3) smaller the protein the further it will travel
4) take polymer page and place onto SDS
5) now polymer will have proteins in same position as on SDS-page
6) place on PVDF membrane on top, filter paper and sponges on either sides
7) add primary antibodies specific to protein of interest
8) add secondary antibody specific to primary antibody (conjugated with reporting system)
9) add substrate
10) If protein present and antibodies bound then signal will occur e.g. visualise on x ray paper

Question 10

protocol for WB

Answer 10

1) add bovine serum albumin to block non-specific sites
2) wash with PBS
3) incubate primary antibodies
4) wash with PBS
5) incubate with secondary antibodies
6) wash PBS
7) visualise signal

Question 11

BSA

Answer 11

bovine serum albumin

Question 12

PBS

Answer 12

phosphate buffer solution