Cell Bio Lab Final

Purpose of Bradford assay

-Measures protein concentration in a solution.

-It relies on Coomassie Brilliant Blue G-250 dye, which binds non-covalently to aromatic and positively charged residues in proteins

Beer Lambert Law

Absorbance (A) is proportional to concentration (c):

A=εlc where:

ε: molar absorptivity

l: path length (cm)

c: concentration (mg/mL)

Linear range: 125-1000 µg/mL for BSA.

General Protocol

1. Prepare a standard curve using known concentrations of BSA.

2. Mix samples with Coomassie Plus reagent (0.95 mL reagent + 0.05 mL sample).

3.Let incubate (optional but preferred for consistency).

4. Measure absorbance at 595 nm.

5. Plot standard curve and interpolate unknowns.

How was data analyzed in Bradford assay

Plot Absorbance vs. Concentration.

Use a line of best fit (linear regression).

Use the equation y=mx+b to find unknown concentrations based on absorbance.

Why did you invert the conical and cuvette rather than shaking or vortexing

Inverting prevents the formation of bubbles/foam while still mixing the solution

Why would incubation for 10 minutes provide more consistent results

Incubation allows the dye-protein complex to stabilize, reducing variability between samples and giving more consistent and reliable absorbance readings.

Higher absorbance in Bradford indicates

Higher concentration of proteins that bound the dye

What wavelength of light was used for BA

595 nm

Do the proteins in sample affect results

yes if there are more basic or aromatic proteins they will have a hgiher absorbance bc stick to dye more

advantages and disadvantages of BA

adv- quick results, highly sensitive to small amounts of protein

disadv- proteins that differ significantly in amino acid sequence from BSA would have different results, surfactant and detergents interfere with results

why is bsa used if its liver and brain extract

BSA is not reactive and is stable but may have similar composition to the enzymes in this extract

2 other protein concentration methods

Lowry assay, Kjdeahl Method

Coomassie dye

Protonated Dye Forms (acidic pH): Pale red or green (λmax = 470–650 nm)

Protein-Bound Dye Form: Blue (λmax = 595 nm)

Binding occurs under acidic conditions and is non-covalent and stable for hours

What does SDS do to proteins in SDS-PAGE?

Make uniform negative charge

Why use β-mercaptoethanol in SDS-PAGE?

Break disulfide bonds

Stacking vs. Separating Gel

Stacking- low % acrylamide, pH ~6.8): Aligns proteins.

Separating- higher % acrylamide, pH ~8.8): Resolves proteins by size.

What's the function of Coomassie dye?

Stains proteins for visibility

Do proteins run to positive or negative end

Positive because negatively charged backbone + SDS

Running buffer solution contains

Tris, glycine, and SDS. Tris has a pKa that functions in as a buffer in the pH range for most biological systems, prevents protease activity. Glycine is an amino acid that is dense and helps the sample proteins sink into the stacking gel.

The sample loading buffer (Laemmli buffer) contains

tris, glycine, SDS, beta mercaptoethanol (BME), and Bromophenol Blue. Bromphenol for visualizing

General protocol for coomassie stain

Prepare Samples: Mix protein with Laemmli buffer (SDS, β-mercaptoethanol, bromphenol blue dye, Tris, glycerol), heat 5 min at 65°C.

Load Gel:Load 30 µL of sample/ladder into wells of a 10% Tris-Glycine gel. Avoid bubbles.

Run Gel:Electrophorese at 60 mA for 45 min (proteins migrate toward the + electrode).

Stain Gel:Wash gel 3× with water (5 min each), stain with Coomassie dye for 20–60 min, visualize bands.

Agarose vs PAGE

Agarose:

For DNA/RNA- separates based on bp length

Horizontal gel

Lower resolution

Uses DNA's natural negative charge

Single gel, TAE/TBE buffer

PAGE:

For proteins- separates based on weight of protein

Vertical gel

High resolution

Uses SDS to add negative charge

Stacking + separating gel, Tris-Glycine buffer kDa

Technique for isolating DNA sequences

Southern Blotting

Restriction enzymes cut DNA fragments separated by gel electrophoresis

Fragments transferred from gel to membrane

Treated with complementary radioactive probe

Detection through x-ray imaging

Technique for isolating RNA sequences

Northern Blot

Denaturation is key in

RNA Samples transferred from gel to membrane

Treated with probe and hybridizes with RNA fragment

X ray imaging for detection

Electrophoresis (Western Blot)

Protein quantification is necessary before loading to ensure proper concentrations and treatments

SDS PAGE for electrophoretic separation

Separation based on size

SDS denatures protein until 1'structure is obtained and distribute negative charge

Proteins travel to positive electrode. "Smallest mass moves fast

Protein transfer

Proteins transferred from gel to membrane (PVDF or NC)• Electrophoretic transfer• "Sandwich" gel with membrane and filter paper

Blocking and incubation

Blocking prevents non-specific binding with antibody. Detection of target protein with primary antibody. Different organisms serum between sample, primary, and secondary antibodies

Direct detection vs indirect detection

direct is faster but labeling is harder and less amplified signal bc no secondary antibody

most antibodies are tagged with __ that react with __

Horseradish peroxidase or alkaline phosphatase that react with chromogen/chemiluminescent substrates

What is the purpose of using trypan blue in a cell count experiment?

To stain dead cells

Trypsin

releases adhered cells from bottom of plate

If you counted an average of 150 cells per grid in a 1:10 dilution, what is the concentration of the original sample?

150 cells/mLx10^4 x10=1.5x10^7 cells/mL

for cell quantity, multiply by volume in mL

adherent vs suspension cells

adherent attaches to bottom of plate suspension floats in media

What does the m6A dot blot assay detect?

N6-methyladenosine (m6A) modifications in RNA or DNA.

What type of membrane is used in the dot blot protocol?

Nitrocellulose NC

What is the purpose of UV crosslinking in the dot blot?

To fix the denatured RNA/DNA onto the membrane.

Why are samples denatured at 95°C before spotting?

to remove RNA secondary structures

What is the role of methylene blue staining?

Serves as a loading control for RNA/DNA.

What is used to detect m6A-specific signals?

HRP-conjugated secondary antibody + ECL (enhanced chemiluminescent) substrate

How are signals from the blot visualized?

Chemiluminescent imaging system (e.g., CCD camera)

Primary cells vs transformed cells

Primary cells - taken directly from living tissue to mimic in vivo

Transformed cells - induced phenotypic alterations due to changes in DNA & gene expression

ATDC5 CELLS (MURINECHONDROCYTES)

Mouse teratocarcinoma cells

Adherent cell line

Media: DMEM

Passage in cell culture

moved to next plate