1
Q
What are the principle steps of affinity chromatography?
A
- Sample introduction/injection
- Adsorption of target molecules
- Wash impurities
- Elution of target molecules
2
Q
What is the affinity resin comprised of?
A
- biospecific ligand
- support matrix
- biochemically inert spacer
3
Q
What are the targets for affinity chromatography and the possible ligands for the targets?
A
Enzyme- substrate, inhibitor, cofactor
Antibody- Antigen
Antigen- Antibody
DNA-complementary base sequence, DNA binding protein
RNA-complementary base sequence, RNA binding protein
4
Q
Look at the chart of the TYPES of fusion Tags
A
…
5
Q
What are some features of the matrix in affinity chromatography?
A
- the material that ligand or spacer is bound to
- it should be rigid, stable, and have a high surface area
- agarose is the most popular cellulose, dextran, and polyacrylamide have been used
- sepharose is a bead-formed of agarose gel
6
Q
What are some features of the spacer in affinity chromatography?
A
-carbon chain interposed between ligand and matrix
-used when active site is located deep within a sample molecule
-if too long, it can interact with sample species on its own (hydrophobic interactions)
if to short, ligand can’t reach active site on sample molecule
-commercial phases have spacers that are optimized of specific seperations
7
Q
What is the purpose of the Matrix?
A
to act as a solid support for the ligand so that it does not just go into solution
and exit the column.
8
Q
What is the purpose of the spacer?
A
to present the ligand so that its important sites are displayed to the
molecule of interest in the sample
9
Q
What is the purpose of the ligand?
A
to bind the molecule of interest in the sample
10
Q
What is the purpose of preparation of homogenate (lysate) before the column.
A
in affinity chromatography the purpose in preparing the homogenate before the column is to lyse cells and release cellular components.
11
Q
what are the steps used in preparation of homogenate (lysate)?
A
- Incubate with lysozyme (in the presence of protease inhibitor)
- Freeze Thaw
- Sonication: high frequency sound waves (ear guards)
12
Q
In the preparation of the homogenate what can be used in place of sanitation?
A
French press
-for large samples
13
Q
What is the purpose of the first step in the prep of the homogenate (lysate)?
A
Incubate with lysozyme (in the presence of protease inhibitor
-lysozyme hydrolyzes bond in bacterial cell wall
14
Q
What is the purpose of the second step in the prep of the homogenate (lysate)?
A
Freeze thaw
-disrupts cell membrane and allows for release of cellular components.
15
Q
What is the purpose of the third step in the prep of the homogenate (lysate)?
A
Sonication
- further disrupts cells and separates proteins from lipids
- shears DNA
16
Q
In affinity chromatography what are the characteristics of the Introduction/injection of sample?
A
the sample generally contains the target molecule along with cellular impurities (other proteins, lipids, DNA, etc.)
-Used a crude homogenate of our clones that were induced for expression of the GST::EGFP fusion protein.
-MUST ensure adequate column capacity
17
Q
In affinity chromatography what are the characteristics of the adsorption of molecules?
A
- use a slow flow (gravity) to drive target molecules toward fresh ligand sites
- The GST (glutathione-S-transferase)::EGFP fusion protein will adsorb to the glutathione present in the affinity matrix
**EGFP is along for the ride, GST has the affinity for the column
18
Q
In affinity chromatography what are the characteristics to wash impurities from the column?
A
- Molecules with no affinity for the ligand are washed from the column.
- Cellular molecules other than the GST::EGFP fusion protein will wash through the column, and the GST::EGFP fusion protein will remain bound in the column.
19
Q
In affinity chromatography, your molecule of interest remains in the column while other molecules are washed out because your molecule of interest….
A
binds specifically to the ligand in the resin while other molecules do not
20
Q
In affinity chromatography what are the characteristics of the elution of target molecules from the column?
A
Elution releases bound protein and also serves to regenerate your column
-The GST::EGFP fusion protein will be eluted from the column using a solution of reduced glutathione
21
Q
What are the Elution methods and what are characteristics of each?
A
Biospecific
- an inhibitor is added to the mobile phase (free ligand)
- the free ligand will compete for the solute
- this approach is often used when low molecular weigh inhibitor is available
Nonspecific
- a reagent is added that denatures the solute (pH, KSCN, urea, ionic strength)
- once denatured, the solute is released from the ligand
- If the solute is to be further used, it must not be irreversibly altered.
22
Q
What if the fusion tag will affect later experiments?
A
?`
23
Q
What are the options for cleavage of the fusion tag?
A
- Cleave off target protein while tag remains bound to resin
- Rather than elute fusion protein, you would wash int buffer compatible for your protease (eg. some proteases need low salt, or no metal ions to work.)
- Flow on buffer containing protease, cap and let sit, eg. room temp 1 hour or 4 Degrees C overnight (be careful of over digestion; there could be a secondary site that is “close enough” to recognition sequence)
- To recover your protein, wash with same buffer lacking protease (do not use “elution” buffer since we want the tag to remain bound to column” - Elute from column and then cleave off tag
- Elute with excess ligand (e.g.. gluathione of GST tag) in an appropriate buffer for protease
- Add protease and incubate (time course experiment would be good)
- Need buffer exchange to remove free ligand in order to bind tag back to column (you want your protein to flow through), or could use a different type of column to separate tag from the target protein.
24
Q
How do you asses the protein purity after affinity chromatography?
A
- Run SDS-PAGE of crude homogenate and fractions
- Stain gel, with GelCode blue or Coomassie (we want to sell all protein so don’t use antibodies)
- For increased clarity use the optional water wash enhancement step (1 hour)
25
In affinity purification, the sample that has the highest amount of total protein is?
Crude Homogenate
26
In affinity purification, the sample that has the highest total amount of the protein of interest is?
Crude Homogenate
27
In affinity purification, the sample that has the lowest amount of protein of interest is?
Wash fraction
28
In affinity purification, what is the sample that where your protein of interest is most pure?
Eluate Fraction
29
What are the methods for quantifying protein?
Lowry
BCA
Bradford
30
How is the protein concentration estimated among Lowry, BCA, Bradford?
For each of these assays, protein concentration is estimated by comparison to a STANDARD CURVE constructed by assaying a series of known concentrations of a reference proteins such as BSA
-absorbance readings must be <1 or samples must be diluted
31
How does the BCA assay work?
Protein reduced Cu+2 to Cu+1 in an alkaline enviroment. The cuprous cation, Cu+1 ion, is chelated and detected using a regain containing bicinchonic acid (BCA). The PURPLE reaction product formed absorbs strongly at 540nm or 560nm, and is linear with increasing protein concentrations between 20ug/mL and 2000ug/mL
32
What is the advantage of BCA assay?
not as much stuff or as many buffers interfere, COMPATIBLE with detergent
33
What are the disadvantages of BCA assay?
DEPENDENT on Tyrosine Residues
34
What are the likely uses of the BCA Assay?
If you have a sample that was prepared using detergent. Use ONLY FOR MIXTURES OF PROTEINS where an “average” amount of tyrosines would be present. Don’t use for pure protein because number of tyrosines can skew results.
35
How does the Bradford Assay Work?
- dye binding assay (Coomassie G-250)
- absorbance max for an acidic solution of Coomassie G-250 shifts from 465nm (brown) to 595nm (blue) in presence of protein.
- Coomassie G-250 in phosphoric acid/methanol is mixed with protein sample and the absorbanc change at 595 nm is proportional to amount of protein in sample
- Detectability 1-1400 ug/mL
36
What is the advantage of using a bradford assay?
dye binds to primarily basic (especially arginine) and aromatic amino acid residues, not dependent on single amino acid
37
What are some stuff that interferes with the Bradford Assay?
Not comparable with DETERGENTS and some common protein buffers
38
What is the likely use for a bradford assay?
can use for pure proteins since not dependent on single amino acid
-CANNOT use if sample was prepared using detergent
39
You have isolated outer-membrane proteins from E. coli using a procedure that takes advantage of differential solubility of membrane proteins in sarkosyl (a detergent). Which assay would you use to determine the protein concentration in your prep?
BCA
40
You’ve just isolated purified BIT:His from an affinity chromatography column in a nonspecific elution buffer (basic). Which assay would you use to determine the protein concentration of your purified protein?
Bradford
41
Sanger DNA sequencing
use dideoxynucleotide (ddNTP):
- missing 3' OH, making it unable to form phosphodiester bond
- Chain terminator
- set up for reactions (one for each ddNTP) add four normal dNTPs along with limiting quantities of the ddNTP
- DNA polymerase will use the template and the one primer to synthesize the complementary strand
- Most of the time the "normal" dNTP will be added. However at some point (randomly) a ddNTP will be incorporated and elongation stops.
- Runs reactions on polyacrylamide gel and "read" sequence" from bottom of film. EITHER THE PRIMER OR DDNTPs are RADIOLABELED, ALLOWING FOR VISUALIZATION ON X-RAY FILM
42
In Sanger DNA sequencing, what stops the DNA chain from elongating?
you add a small amount of dideoxynucleotide
43
If you would like to sequence the junction between the 5' fusion tag on your vector and your cloned insert, which would you add to the sequencing reaction?
Forward primer
44
Next generation/deep/pyro/ high throughput sequencing
-The human genome project and genomes of several model organisms were produced largely by conventional Sanger-based capillary sequencing
-Starting in 2005, new platforms appeared
-Pyro-sequencing (next generation/deep/high throughput sequencing) relies on
detection of pyrophosphate release on nucleoside incorporation, rather than chain termination with ddNTPs (“sequencing by synthesis”)-Can obtain only 500 bp reads, but can obtain 400 million nucleotides of data in a 10 hour run.
- Expensive, but getting cheaper!
-Commonly used for sequencing genomes and transcriptomes, but not plasmids (used in place of microarrays)
-You must generate library of DNA fragments and from there each platform will
vary in terms of how sequencing is performed.
45
Look at chart of comparison of sequencing platforms?
?
46
Genome sequencing
to characterize an entire genome of any size/complexity; can be re-sequencing or denovo sequencing
47
Exome sequencing
to sequence part of a genome through enrichment strategies to target coding regions; enables the identification of single-nucleotides variants and small insertions or deletions
48
transcriptome sequencing or RNA-seq
to identify and quantify RNA, including differential gene expression, identification of alternative splicing, and gene discovery
49
What is the future of sequencing?
Nanopore sensing
- analyte passes through the pore or near its aperature
- This event creates a characteristic disruption in current
- by measuring that current, its possible to identify the molecule in question
- This system can be used to distinguish between the four standard DNA bases as well as base modifications
50
What does SDS page determine?
-determines gel migration by protein molecular weight
51
What are some applications of SDS page?
- Size determination
- Estimating Purity
- Estimating levels of expression
- Immunoblotting (Western Blot)
- Preparing for protein sequencing/mass spec
- Generating antibodies
52
DNA vs. Protein Gel Electrophoresis
Similarities:
- separate molecules by size
- utilize an electrical charge to move the molecule through the buffer
- Require a matrix with a small enough pore-size to separate molecules
Difference:
- DNA is run on agarose gel, proteins on polyacrylamide gels
- Proteins are smaller than DNA
- polyacrylamide matrices have a smaller pore size than agarose
- DNA is exception: very short DNA molecules are run on polyacrylamide gels
- DNA is run at constant voltage and protein is run at constant current
- ALL DNA is negatively charged, but protein have varying charges depending on amino acid content of the specific polypeptide and buffer pH (proteins are positive some negative)
- DNA has little secondary structure while proteins can be highly folded
53
SDS PAGE characteristics
- allows protein to migrate by size alone
- loading buffer is added to all samples and then boiled prior to loading onto the gel. Loading buffer for SDS page contains:
1. Glycerol-helps samples to sink to bottom of the well
2. Bromophenol blue-tracking dye
3. B-mercaptoethanol (BME)
- SDS is an anionic detergent that denatures proteins by wrapping around the polypeptide backbone (forming a micelle), and thus confers a net negative charge in proportion to polypeptide length.
- SDS disrupts most non-covalent bonds, decreasing protein folding
- A reducing agent (B-mercaptoethanol or dithiothreitol) is added to reduce cysteine bonds (disulfide bonds) and "unfold" proteins.
- After boiling in SDS and BME, all proteins act as negatively charged linear molecules and can be electrophoretically separated by size.
54
In SDS Page, what chemical is used to ensure that all protein molecules are coated with negative charge?
SDS
55
In SDS-Page, what chemical is used to ensure that protein disulfide bonds are broken?
BME
56
In the discontinuous gel system what does the stacking portion do?
- proteins get "stuck" between the mobility of the buffer ion of the same charge (usually negative charge) in the stacking gel (leading ion) and the mobility of the buffer ion in the upper tank (trailing ion)
- When electrophoresis is started, the ions and the proteins begin migrating into the stacking gel.
- The proteins concentrate is a very thin zone, called the Stack between the leading ion and the trailing ion
- The proteins continue to migrate in the stack unit they reach the separating gel
57
In the discontinuous gel system what is the purpose of the resolving gel?
a pH or an ion change occurs when the proteins reach the resolving gel, and the proteins become the trailing ion and "unstack" as they separate on the gel.
-portion of the gel where the protein bands separate by size (or resolve)
58
What is the unique advantage of running a discontinuous protein gel rather than a continuous?
better resolution of the protein bands
59
What it the advantage of treating protein with SDS and b-mercaptoethanol?
Because proteins can be separated by size alone, rather than being dependent on size, shape, and charge
60
What are the two methods for visualizing proteins?
Nonspecific- stain the gel itself with either coomassie blue (or Gelcode blue) or silver stain to see all proteins on gel
-can do a temporary nonspecific stain with water soluble dye(ponceau red) in order to see total protein profile.
Specific- transfer all proteins to nitrocellulose (or PVDF) membrane for western blot in order to probe for specific proteins
61
How do you estimate protein size?
assume average weight per amino acid=114 Da.
EX:
gst=660 bp, encoding 220 aa=25080 Da
egfp=720 bp, encoding 240 aa=27360Da
Weight of fusion=52,440Da or 52kD
62
Your fusion protein should appear as an intense band slightly larger than 52 kD in your positive clones. You may see an intense band of about half that size in your negative clones. Where does this band come from?
GST
63
What does protein detection with antibody confirm?
confirms expression of specific protein, and therefore can infer presence, orientation, and easing frame of DNA insert
64
How do you go about making an antibody for a specific protein?
- full length native protein
| - peptide
65
Full length vs peptide
full length
- likely to efficiently recognize native structure of protein
- Hard to ensure specifically against other structurally related proteins
- Not always easy to purify/obtain
peptide:
- dont need to be able to make full length protein
- Enhanced specificity against desired protein (different antibodies can be made to different isoforms)
- simply purification of antibody
- doesn't always work, and REALLY doesn't always work for detection of endogenous protein
66
how is the production of polyclonal antibody done?
1. animal selection-choose animal based on the amount of antibody needed
2. Inject the antigen into the animal
3.antigen activated B cells
4a) can make memory B cells
4b) can make plasma cells
5B) secretes polyclonal antibodies from different B cells
6B) serum containing polyclonal antibodies is harvested from the animal
67
Secondary antibody fragments
-reduced non-specific binding, particularly in immunohistochemistry and flow cytometry experiments where Fc receptor may be expressed
68
antisense strand
used as a template to make a copy of the sense strand.
69
sense strand
used as a template to make a copy of the antisense strand
70
Anchored PCR
one primer anneals to insert
| other primer anneals to vector sequence
71
What would the results be if you had the wrong insert?
?
72
What does the forward primer anneal to?
vector sequence (gst)
73
What does the reverse primer anneal to?
to sequence in egfp
74
What would the results be if you had an *extra* insert (not egfp) between the gst gene and the egfp gene? (assuming a long extension time)
A PCR product larger than the expected size for egfp sequence insertion along
75
What would the results be if you had an *extra* insert (not egfp) at the 3' end of egfp (given that egfp is in the correct orientation)?
A PCR product exactly the same size for egfp insertion alone
76
What will we see for positive clones (with egfp insert) versus negative (vector only, or vector with wrong insert) clones on IPTG plates?
Positive clones will be green, and negative will be normal (e. coli color)
77
What does restriction mapping determine? and why do we want to determine this?
Determine the ORDER of specific restriction sites (and the distances between them) along the length of a DNA molecule by analyzing the sizes of restriction fragments in an agarose gel.
- to determine the existence and orientation of insert DNA (was a cloning procedure successful?).
- to confirm the identity of the plasmids
78
What are the two types of databases?
Primary Database (genbank)
-Original submission by experimentalists
o Database staff review and may organize data, but they
don’t add/modify additional information
o Records are “owned” and updated by their authors
Derivative Database (refseq)
o Human-curated (compilation and correction of data) o Computationally-derived
o Combination
79
Who can submit to Genbank?
ANYONE
80
GenBank
-Nucleotide sequence database for all known sequences
-Each DNA record has a unique identifier (accession number)
-You can use GenBank to find your gene or protein sequence of interest. You can enter an accession number, key word, gene name, etc.
o But remember to take the info with a grain of salt; GenBank entries can have mistakes (your Grandma can enter a sequence if she wants!)
81
RefSeq
RefSeq sub-database is NCBI-curated
They take GenBank files and try to compile the best info
• Forming the “best representative” sequence
• Standardizing nomenclature and record structure
• Adding annotation (references, sequence features)
• Standard accession numbers
- NM_# - NC_# - NP_#
mRNA
complete chromosome or genome protein
Refseq files can still have potential errors, but are more reliable than regular GenBank files.
• The disadvantage of RefSeq is that NCBI curators cannot keep up with all
nucleotides sequences, so not every gene is represented.
• Use it when you can, but if you don’t find what you need, use the GenBank
database.

82
Benefits of Refseq
-Non-redundant
• Explicitly linked nucleotide and protein sequences
• Updated to reflect current sequence data and biology • Usually validated by hand (can still have errors)
• Format consistency
• Distinct accession series(withanunderscore)
- http://www.ncbi.nlm.nih.gov/RefSeq/key.html#accessions • Stewardship by NCBI and collaborators
83
Blast searches
Basic Local Alignment Search Tool
| -Compare your favorite nucleotide or protein sequence to a database of all known sequence
84
nucleotide blast
Search a nucleotide database using a nucleotide query
85
protein blast
Search protein database using
| a protein query
86
blastx
Search protein database using a translated nucleotide query
87
tblastn
Search translated nucleotide database using a protein query
88
tblastx
Search translated nucleotide database using a translated nucleotide query
BIT 410
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