Exam 2 Gene Expression/Western Blot

Why do we want gDNA?

  • want to study the Promoter region

  • gDNA has all the necessary noncoding and going regions i.e. exon and introns

Gene Expression Analysis:

  • Northern Blot

  • RT-qPCR

~~RT-qPCR

  • produce more PD2 (proliferated) and DD7 (differentiated)

~SYBR Green Supermix (2x rxn)

  • Amplify

    1. Taq Polymerase (antibody-mediated)

    2. Buffer

    3. DNTPs

    4. MaCl2

    5. RT rxn (template)

    6. Primers (forward and reverse)

  • Quantity

    1. SYBR Green (fluorescent)

    2. Enhancer and Stabilizer

    3. ROX passive dye

SYBR Green

  • will bind to dsDNA causing fluorescents

Taq Polymerase (antibody-mediated)

  • prevents Taq from binding till needed

  • the antibody will denature till 95C

  • the antibody binds to protein to prevent rxn from occurring

ROX passive dye

  • baseline Ct reading will not change

Enhancer and Silencer

  • transcription factors that bind to sequences in the promoter region

  • TAA derivatives

  • prevent primers from associating to SYBR Green

Cross Ct- line (cycle threshold)

  • the sooner the Ct-line is passed the more inial RNA we have

    • gene expression is higher

  • the longer it takes to pass the Ct-line the lesser inial RNA we have

    • gene expression is lower

Platue

  • All SYBR Green is bound

Melt curve

  • The longer it takes, the longer the amplicon

  • The shorter it takes, the shorter the amplicon

  • two peaks for one, possible contamination

  • Two peaks from two means one is short one is long, better.

~~Primers

  • 18 - 22 bp

  • 75 - 150bp (amplicon)

  • GC content (40% - 60%)

  • both GC have to be around the same content

  • Cross Exon Exon junction

Primers need to avoid gDNA contamination to eliminate the impact

Exon Exon junction

  • keeps a Short amplicon,

  • lowers the chance of contamination

  • end with AG - that is what the spliceosome recognizes

AG/ GU rule

  • Cuts end then covalently lariad structure that it releases

*Can put primers on certain exons to distinguish between multiple possible spice variants going on.

Alternative Splicing:

  • Primers can be placed on certain exons to distinguish between multiple possible splicing variants

~~DNA isolation (RT-qPCR)

GAPDH

  • reference gene

  • GAPDH gene and Gene of interest should be the same or close numerically when measured

  • The reference gene is used to correct for variation in biological replicates if something went wrong with RNA preps.

  • Reference genes tell us if variation in biological replicates is because of something normal for that gene or because of something that went wrong with the RNA preps. If the biological replicates and reference genes go up the same way, then it means the RNA preps were not good.

~~Promoter Region

  • used to study how a gene is regulated

    • RT-qPCR can not do this

~Isolate Promoter: (PCR)

-promoter DNA is gDNA that has the sequence we will PCR amplify

  1. Purify gDNA

    (Look in the database for sequence)

  2. Create primers

  3. PCR

    1. Use primers to amplify fragments of the promoter of interest based on the promoter sequence

  4. Engineer restriction digest (identify where plasmids go)

  5. Insert into a reporter plasmid

How to clone promoter region?

  • PCR

Process:

  1. Purify PCR product

  2. Digest

  3. Ligate into reporter plasmid

  4. Transformation and inoculate

  5. Mini - prep

  6. Run gel

_________________________________________________________________________________

Reporters do not come w/ promoters

  • Need to design primers to PCR amplify

________________________________________________________________________________

Review

PCR amplify fragments of the gene promoter

What enzymes do we need to cut?

  • Restriction digest

    • Forward primer and reverse primer

    • needed to amplify both strains of dsDNA

  • Marks the region you want to amplify

~Reverse primer binds to the template (near the first Exon)

~Forward primer binds to the complementary

Why multiple fragment sizes?

  • We do not know what is in the sequence in the regulatory regions

What is the problem of taking the regulatory region from one gene and ligating to another gene?

  • There is always the possibility of missing a regulatory element

____________________________________________________________________________________

The regulatory region

  • PSEAP-2

    • Reporter plasmid

  1. Amplify Reporter plasmid w/ PCR

  2. Analyze sequence -UBR 5

    1. Design Primes

      1. GC content

      2. length

      3. *Do not cut DNA within in sequence. hjhjbjhbjh

      4. *Recognize the sequence of plasmid we want to clone into reporter.

  3. Transfect into cells

NEB cutter sequence

  • mlu 1

Cutter Sequence needs to be in the MCS

  1. PCR -digest

  2. Analysis

  3. transfect

Trasfect - introduce recombination gene into the cell, gene will activate transcription

  • The reporter gene should transcribe

____________________________________________________________________________________

Restriction digest analysis:

  • Multi fragment sizes

    • differences in the fragment sizes narrow where regulatory elements exist

  • look at enzymes,

    • cut reporter plasmids MCS,

    • cut primers,

    • should not cut gDNA

Get recombinant plasmid: (look at chart)

  • Trasfect into cells

    • Introduce DNA into Euk cells

  1. Recomb gene transcriptionally active

  2. The reporter gene should be transcriptionally active

  3. Cells make a lot of SEAP protein in growth media

SEAP protein (if transfected will transcribe causing light)

  1. transfect

  2. media

  3. Add SEAP substrate (heat up to 65C to denature phosphatase)

  4. Produce light

  5. measure amount of light (RLU)

Enhancers:

  • Elements unique to the promoter that cause it to be responsive to the same conditions.

  1. Canonical

    1. response element sequence

  1. MRFs (myogenic regulatory factors)

    1. Myogenin

    2. MyoD

  1. Proteins that regulate transcription

  2. Only capable of binding DNA to response element with certain seq of nucleotides

Bind to the enhancer sequence called E-box.

  • E-box location in the promoter

    • 5` CAN N TG `3

Genes w/ E-box can respond to MRFs and expression

Clone fragment into receptor Plasmind:

  1. Run SEAP

  2. Identify potential E-box sequence

What do we expect the promoter to do?

  • MRF should drive RLU higher if you have MRF present along with the reporter gene.

Site-directed Mutagenesis: (look at charts)

  • Reporter plasmid with SEAP gene and promoter with E-box

Forward Primer

  • Spans across E-box, change

    • Change 2 nucleotides in the primer

      • (Everything else is the same as a primer will bind.)

Reverse Primer

  • The exact same complementary sequence

  • Primers become part of PCR product

~Find template Plasmid vs. PCR amplified Plasmind?

  • Template Plasmid was grown in the presence of bacterial cells

  • Bacteria methylates DNA in certain places.

Use Enzymes Dpn1 to digest rxn afterward

  • only recognizes 4bp sequence that has to be Methylated

    • Cuts only the template DNA into fragments

  • PCR product DNA is left behind

How do you find the Plasmids that you want in a mixture?

Transformation

  • Bacteria takes up mutagenic plasmid

**Western Blot**

~~ ULB +

Lysosomes have lots of proteases. We want to protect proteins as we lyse cells.

Lysosomes will not work when pH around 5.

  1. Protease Inhibitors 2. Phosphatase inhibitors

  • 1. Protease Inhibitors: Cocktail

    • Aptoninin: Serine protease inhibitor

    • Chymastatine: Serine and Cysteine protease inhibitor

    • Leupeptin: Serine and Cysteine protease inhibitor

    • Pepstain: general Inhibitor (blocks lots of Proteases)

  • 2. Phosphatase inhibitors: Cell signaling

    • Beta- glycerophotase *

    • Sodium molybdate *

    • Sodium othoranadate *

    • *main ones*

    • DMSF

  • 3. DTT : Breaks disulfide bridges

Make ULB (Lysis Buffer)

  • SDS

  • Tris

  • NaCl

  • NaF pH inhibitor

  • DTT

Do 5’ fold and 10’ fold dilutions with lysis buffer and do reading of those

  • whichever one gives you reading within the linear range of the assay is one you’ll use.

*Western Blot*

RNA —→ Protein

Translation —> Western Blot

  1. Isolate Protein from tissue or cells

  2. Lyse cells — make homogenate

Basis* ULB

Lysis Buffer (ULB):

  • 1. Detergent: (SDS) non-ionic detergent*

    • NP-40

    • Triton X100

    • 5% w/v

  • 2. Tris: pH regulator*

    • pH 7.5

    • 50 mM

  • 3. NaCl (salt) - 50 mM*

    • Dissolves into ionic form when mixed with H2O

      • Change the salinity of the solution around the cell

      • create hypotonic solution

      • lots of ions inside cells

      • Water rushes in, destabilizing the membrane

    • Lyses cell

  • 4. NaF: Phosphate inhibitor - 50mM

Make Buffer

  • Weight out solids

  • Add water (amt. based on solvent)

  • Add HCl to bring pH down to 7.5

  • Add water

Protein Assay:

Protein Assay: Bradford Assay

  • Commassile Brilliant blue G - 250 dye

    • Dye binds to basic amino acids arginine and aromatic a.a

      • changes to blue

    • More protein you have, more dye that binds, more blue you get

  • If we do lysis and have concentrated protein lysate we’ll have to serail dilute a couple of times.

_____________________________________________________________________________

  • y=mx+b x is mult by 2 or 5.

  • look at notes for math portion

______________________________________________________________________________

SDS-Page: (gel electrophoresed for proteins)

  • Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (8% -12%)

    • anionic detergent

  • Coats proteins

    • uniform negate charge

Acrylamide

  • thin, run up and down, separate proteins.

Glycine

  • an amino acid that can be protonated or deprotonated

TEMED

  • the catalyst causes the cross-pattern

APS

  • catalyst polymerizes acrylamide

Loading dye for SDS-PAGE

  1. Mix diluted proteins with loading dye

    1. Tris Buffer -7.5

    2. Bromophenol Blue

    3. Glycerol

    4. DTT

    5. Glycine

Gel components: what do they do?

Tris pH 6.8 standing

Tris ph 8.8 running

  1. Glycine

  2. SDS

  3. Acrylamide

  4. Bis - acrylamide

  5. APS

  6. TEMED

  7. H2O

Running buffer

  1. SDS

  2. Glycine

  3. Tris

Shape- linear

  • denature 95C for 5 min

  • Breaks disulfide bridges (DTT or Beta- mercaptoethanol)

    • Reducing agents

~~Transfer

  • Buffer

    • Glycine

    • Tris

Structure for gel stain

Metal plate

filter paper

PVDF membrane (methanol)

Gel

Filter paper

Metal plate

  • PVDF has the transfer

Stain membrane with

  • Ponceau S (red dye)

    • protein adhesión

  • Wash H2O

Block with milk in T-TBS

  • Clean off any weakly associated bonds

    • Saline weak ion and bonding

    • weak hydrophobic

Process:

  1. Milk - fill all open spaces on the membrane

  2. Wash T-TBS

    1. 3x -5min

  3. Incubate with 1` antibody diluted with MyHC

    1. in 5% milk + T-TBS (10mL)

    2. 1-2hr @RT

  4. Wash T-TBS

    1. 3x -5min

  5. Incubate with 2` antibody

    1. 5% milk + T-TBS (30-60 min)

  6. Wash T-TBS

    1. 3x -5min

  7. Develop

    1. light production

What is the purpose of using a secondary antibody for western blotting?

  • Bind to the primary antibody, Amplify the level of the detection signal, and Eliminate the need to enzyme-conjugate every primary antibody.