Detecting and Quantifying Proteins

Question 1

does more RNA equal more protein

Answer 1

no

Question 2

SDS

Answer 2

Sodium Dodecyl Sulfate - detergent made to denature proteins and make them uniformly negative

Question 3

PAGE

Answer 3

polyacrylamide gel electrophoresis (alternative to agarose, better for proteins, small proteins travel through the gel faster, ladder including fragments of known size used to estimate size of sample proteins

Question 4

Antibodies

Answer 4

Proteins that bind specific peptides with high affinity, can be made in animals by injecting them with forein proteins/peptides

Question 5

MHC

Answer 5

major histone compatability, displays peptides from each cell to the immune system (8-10 aa each resulting from degradation of proteins in the cell

Question 6

T cell receptor

Answer 6

ummunoglobin checks it’s not a self protein, sets combination of VDJ segments that make variable region that wont react to self proteins

Question 7

immune cells

Answer 7

produce antibodies that recognize non-self proteins

Question 8

How do we detect specific proteins

Answer 8

proteins don’t form base pairs but they can be recognized by antibodies,`

Question 9

Immunoprecipitation

Answer 9

Let’s us isolate proteins and their binding partners. Start with a protein complec , add a primary antibody, a secondary antibody thats apart of an agarose bead, the protein complex is washed away, put it through an elution (high pH, and then perform SDS-PAGE or Western Blotting)

Question 10

Cell-resolution analysis in formaldehyde-preserved (fixed) cells

Answer 10

target protein recognized by primary antibody (ab), primary ab recognized by flurescent secondary ab, and it is visualized with fluorescence microscopy

Question 11

RNA silencing

Answer 11

mRNA hairpins made in nucleus, hairpin removed by dicer, mature miRNA loaded into RSIC complex that guides silencing or destruction of complementary sequences

Question 12

RISC

Answer 12

RNA Induced Silencing complex

Question 13

Imaging in tissues and cells

Answer 13

gives information about anatomical and subcellular localization

Question 14

Oct4

Answer 14

pluripotent stem cells

Question 15

Cdx2

Answer 15

cells that will form placenta

Question 16

Pluripotent cells

Answer 16

are located in the inner cell mass of the plastocyst

Question 17

how to quantify or sort fluorescent cells

Answer 17

individual antibody-labeled cells pass by a laser beam, and are measured for each cell (flow cytometry or FACS)

Question 18

FACS

Answer 18

fluorescence-activated cell sorting

Question 19

pre genomics

Answer 19

targets had to be known in advance (northern blot required complementary probe to detect RNA of interest, qPCR requires primers to detect transcripts of interest, Immunolabelling requires antibodies targeting proteins of interest

Question 20

epigenomics

Answer 20

allows us to map and measure dynamic changes in gene expression, transcription factor binding, and chromatin dynamics during development

Question 21

Next generation sequencing

Answer 21

allows unbiased interrogation of all expressed sequenced, generates millions of short sequence reads (100nt)

Question 22

NGS reads

Answer 22

align reads to genome and quantify number mapping to each exon of each gene, it’s totally unbiased and exon-level analysis, provides information on transcript levels in different samples or conditions, along with information on exon usage and promoter usage, allows us to find needles in the genomic haystack

Question 23

ChIP

Answer 23

Chromatin immunoprecipitation testes interactions between DNA and specific proteins

Question 24

ChIP step 1

Answer 24

treat with formaldehyde to covalently link protein DNA interactions (keeps them from drifting apart

Question 25

ChIP step 2

Answer 25

fragmentation, then antibodies are used to isolate any proteins of interst (immunoprecipitation) along with any DNA that the protein is associated with

Question 26

ChIP step 3

Answer 26

DNA purification, DNA bound to specific proteins can be quantified by PCR or sequencing

Question 27

enhancers

Answer 27

contain binding sites for multiple transcription factors, which must be activated at the same time

Question 28

transcription factors

Answer 28

recruit co-activators that acetylate and methylate local histones (H3K27ac and H3K4me1)

Question 29

Stable TF binding

Answer 29

leads to nucleosome depletion and DNA being "open" or "accessible which allows global identification by sequencing

Question 30

ChIP-seq genome wide analysis

Answer 30

ChIP with indicated antibodies; preform ChIP -seq and RNA-seq ,Map reads to the mouse genome, height of peakes indicate number of reads that map to the location

Question 31

H3K4me1

Answer 31

marks all enhancers

Question 32

H3K27ac

Answer 32

marks active enhancers

Question 33

ATAC-seq

Answer 33

Assay for transposase accessible chromatin, Tn5 transposase inserts DNA into accessible chromatin (enhancers) Inserted DNA servers can be mapped and quantified by NGS, results correlate with H3K27ac ChIP-seq> Allows identification of active enhancers in isolated cells (even sing cells)