explain d1&2 domain on pAlcam, dNAlcam, and sAlcam cells
pAlcam: wildtype, no domains are disrupted, expressing normal Alcam, ALCAM molecules oligomerizes on the cell membrane and thereby create at least two binding locations for opposing ALCAM molecules from another cell. The binding of multiple ALCAM molecules creates a so called “ALCAM network” between cells, thereby mediating cell clustering.
dnAlcam: d1&2 domain deleted, lack of the N-terminal part these mutants cannot engage in binding to other ALCAM molecules
sAlcam: only expressing d1 domain (soluble), also unable to bind to other ALCAM molecules
how many expected cells in a 100% confluency T75 flask?
7.5 * 10^6 cells
(= 100.000 cells / cm2 )
explain ALCAM molecule
- Member of immunoglobulin superfamily
- Two V-type Ig domains, three C-type domains (extracellular)
- Heterophilic (CD6) and homophilic (ALCAM-ALCAM)
interaction
D1-D2-d3-d4-d5-transmembrane protein-intracellular protein
explain MMP2 activation pathway
- integrin will bind to ECM (collagen)
- furin will be expressed by the nucleus causing MT1-MMP14 to form a dimer
- TIMP2 will bind to the dimer
- proMMP2 will bind to the activation complex (MT1-MMP + TIMP2) and cut the pro structure and activate MMP2
3d culture protocol
- collect appropriate amount of cells
- cast cell free collagen layer
- add cells with collagen
- cells will grow in spheroid
- after 24/48h remove D10 medium and replace with D0 medium
- incubate for 24/48h
- collect D0 medium (should contain MMP2)
gene expression analysis flowchart
- total RNA isolation
*really unstable
*in gel electrophoresis should show 28S, 18S and 5,8S bands
*smears? forgot to preheat to 70C (return to secondary structure) or RNA already degraded -> redo - cDNA synthesis
*make the RNA into cDNA -> more stable
*use oligodT to reverse transcriptase mRNAs (bind to their polyA tail)
*use random hexamers to bind to the total RNA
*use specific primer to bind to target RNA - qPCR
calculate annealing temp
T = 81,5 + 0,41(%GC) - (675/N)
zymography protocol
- Sample Preparation: Samples are prepared in a non-reducing loading buffer for SDS-PAGE.
- Electrophoresis: Samples are run on a polyacrylamide gel containing the substrate (e.g., gelatin or casein).
- Renaturation: The gel is incubated in a detergent solution (e.g., Triton X-100) to remove SDS and allow the enzymes to renature.
- Incubation: The gel is incubated in a suitable buffer at an optimal temperature to allow enzyme activity.
- Staining: The gel is stained with a dye (e.g., Coomassie Brilliant Blue) to visualize areas of substrate degradation as clear bands against a dark background
zymography reagent =+control
- Polyacrylamide Gel: Provides a matrix for separating proteins based on size.
- Substrate (e.g., Gelatin, Casein): Embedded in the gel to be degraded by the enzyme.
- SDS (Sodium Dodecyl Sulfate): Denatures proteins and provides a uniform negative charge.
- Triton X-100: Removes SDS from the gel, allowing enzymes to renature.
- Incubation Buffer: Provides optimal conditions (pH, ions) for enzyme activity.
- Staining Dye (e.g., Coomassie Brilliant Blue): Stains the gel to visualize areas of enzyme activity
control: D10, D0 (neg), trypsin
why preheat RNA?
denature to prevent secondary structure
rev. transcriptase primers
OligodTs: 6 T primer, only binds to mRNA, not suitable when RNA quality isn’t good since degraded RNA will have its poly A tail degraded first
random hexamers: 6 random nucleotide primers, binds to the whole RNA sample
how to prevent gDNA contamination in RNA isolation?
rDNAse treatment
cDNA synthesis gel electrophoresis controls
- Positive Control: This involves running an aliquot of intact RNA to ensure that the gel electrophoresis process is working correctly and to rule out any unusual results due to gel artifacts1.
- Negative Control: This typically includes a sample where no reverse transcriptase was added during cDNA synthesis. This control helps to identify any contamination or non-specific amplification that might have occurred.
- DNA Ladder: A DNA ladder or marker is used to determine the size of the cDNA fragments. It provides a reference for comparing the bands observed in your samples2.
- No Template Control (NTC): This control contains all the components of the reaction except the RNA template. It helps to check for any contamination in the reagents or the environment
- No RT (reverse transcriptase): To ensure there’s no genomic DNA (gDNA) contamination in your cDNA synthesis, you should use the No Reverse Transcriptase Control (No-RT Control). This control involves setting up a reaction that includes all the components except the reverse transcriptase enzyme. If you observe any amplification in this control, it indicates the presence of gDNA contamination.
