in vitro experimentation; proteins, DNA-proteins interactions

Question 1

how can we separate proteins?

Answer 1

using SDS-PAGE

Question 2

how can we detect proteins?

Answer 2

using western blot, immunofluorescence, immunohistochemistry, ELISA and FACS

Question 3

how can we study protein complex interactions?

Answer 3

by using co-immunoprecipitation

Question 4

how can we study DNA-protein interactions?

Answer 4

by using luciferase assats, EMSA and ChIP

Question 5

priniciple of SDS-PAGE

Answer 5

separation of proteins based on the molecular weight

Question 6

principle of Western Blot, ELISA, IHC, IF and FACS

Answer 6

detection of proteins using specific antibodies

Question 7

what is SDS-PAGE?

Answer 7

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

Question 8

general process of SDS-PAGE

Answer 8

• proteins are negatively charged (due to SDS) and move to positive electrode
• proteins separate by size (molecular weight)
• smaller proteins move faster

Question 9

what is needed to carry out an SDS-PAGE?

Answer 9

• acrylamide gel
• protein sample
• loading dye (density + dye)
• SDS-based solution

Question 10

how can we identify the proteins when we remove the gel from the tank?

Answer 10

using a dye of Coomassie Blue Stained

Question 11

using antibodies to detect proteins

Answer 11

• antibodies vary in specificity but ideally they only recognise one epitope

Question 12

applications for antibodies

Answer 12

basic research, diagnostics, therapeutics

Question 13

process of western blotting

Answer 13

• isolate all proteins form sample
• gel electrophoresis
• transfer to membrane
• block
• primary antibody
• secondary antibody

Question 14

three ways of protein detection in western blot

Answer 14

1. colorimetric
2. chemiluminescence
3. fluorescence

Question 15

what is the ELISA assay?

Answer 15

enzyme-linked immunabsorbent assay

Question 16

what are the 4 types of the ELISA assay?

Answer 16

• direct ELISA
• indirect ELISA
• sandwhich ELISA
• competitive ELISA

Question 17

what are FACS?

Answer 17

fluorescence-activated cell sorting

Question 18

prinicples of FACS

Answer 18

(single cell analysis)
- specialised type of flow cytometry
- provides method for sorting heterogenous mixture of biological cells into two or more containeers, one cell at atime, based upon the specific light scattering and fluorescent characteristics of each cell
- allows single cell separation and recovery

Question 19

what is co-immunoprecipitation?

Answer 19

a protein complex that can be isolated from a protein mixture by using an antibody that is specific for one protein of the complex

Question 20

what is luciferase reporter assay used for?

Answer 20

for transcription factor activity

Question 21

what is EMSA used for?

Answer 21

electrophoretic mobility shift assay
- determines if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence

Question 22

what is ChIP-Seq used for?

Answer 22

combines chromatin immunoprecipitation (ChIP) with a massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins

Question 23

luciferase reporter vector

Answer 23

sequence of luciferase protein= emits light
- introduction of promoter, if active = transcrip/trans of luciferase protein = light
light measured in luminometer

Question 24

reaction components of EMSA

Answer 24

• protein
• specific competitor
• mutant/non competitor
• probe
• antibody

Question 25

process of ChIP-seq

Answer 25

1. TFs bound to DNA in nucleus
2. fix the protein to the DNA
3. immunoprecipitate the TF of interest, along with the attached DNA
4. sequence the DNA to determine the binding site

Question 26

summary of protein analysis techniques

Answer 26

• antibodies very useful in lab for identification of proteins in WB/ELISA (cell lysates), immunofluorescence/FACS (cells in culture) and immunohistochemistry (tissues)
• protein complexes can be isolated from a protein mix. by using antibody speciic for one protein of the complex (co-immunoprecipitation)
• DNA-protein interactions are critical for gene expression regulation & we can study them with techniques such as luciferase assay, EMSA, and ChIP-Seq