immuneprecipitation for trove transcript variant and western plot

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Paper Format: Number of pages: Type of work: Type of paper: Abstract page: Sources needed

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Yes 8
Subject Biology Topic immuneprecipitation for trove transcript variant and western plot
Academic Level: Professional

Paper details

please see the word fills.

These are the gel pic of immunoprecipitation and western plot of my samples in three type
of imaging.
Lane1: Ladder
Lane2: Antibody (IP)
Lane3: Beads
Lane4: depleted input
Lane5: Input
This is the troubleshooting for the upper image with positive control in the first
4 lanes.
A
B
C
Practical: Immunoprecipation of
TROVE2 protein isoforms from human
cell line protein extracts
Introduction
Immunoprecipitation (IP) assays are extensively used in research to ‘precipitate’ specific
proteins from complex solutions. Western blot (WB) analysis is often used to detect
and compare the presence of ‘specific’ proteins. In the practical you will perform you will use
a ‘specific’ anti-TROVE2 monoclonal antibody to IP and then western blot
to analyse proteins precipitated from a set of human cell line protein extracts. You will
compare the WB banding patterns obtained from a specific extract, to determine which
TROVE2 protein isoforms have been immunoprecipitated, and/or if any, bands are ‘missing’
from a TROVE2 targeted CRISPr genome edited clone cell line.
Below is an overview to the basics of the techniques we will be using and the background to
the cell lines. The actual experimental protocols we will use are outlined in the Practical:
Laboratory work MSE-4041 note
Immunoprecipation (IP) – the basics
Immunoprecipitation (IP) is a method of ‘purifying’ a ‘specific’ protein from a complex
protein mixture. IP uses an antibody, which is specific for the protein. In the first stage of the
IP this antibody is ‘bound’, by it’s constant region – heavy chain, to an agent that will allow
it’s ‘precipitation’. In many cases, this agent is ‘Protein A and/or Protein G’ – which has been
conjugated to tiny magnetic beads. See figure below.See this page from ThermonFisher for
quite a good overview of the IP process using magnetic beads
Western blotting – the basics
SDS-PAGE
SDS-PAGE gels can be used to ‘separate’ and determine the apparent molecular weights of
proteins: different percentages of acrylamide are used to separate proteins of various
molecular weight ranges (see Figure 1).
Western blot probing of protein immobilised on ‘membranes’
Transferring proteins from gels to membranes: this process utilises the fact that the
proteins in the gel will be complexed with negatively charged SDS molecules. As a result,
when placed in an electrical field in a suitable ‘transfer buffer’, the proteins will ‘migrate’ to
the positive anode. If a protein binding membrane is placed in front of the anode, and the
transfer current/buffer correctly chosen, the proteins will be restrained by and adhere to
the membrane. This process can be carried out using ‘Semidry, standardised transfer’ settings and conditions, which is what we will do in the practical.
Watch this video for an overview:
Primary antibody binding: recognize and bind to ’specific’ epitopes. In a western
blot the epitopes available for binding maybe different to those available to binding using IP can you think why (review your lectures).
Secondary antibodies: recognize and bind to primary antibodies. They are ‘manufactured’
by firstly ‘immunising’ one species of animal with immunogenic peptides derived from the
antibodies of the ‘target’ species. This works because the sections of antibodies chosen to
be immunised are ‘different’ enough from the host animal antibodies that they will provoke an
immune response in an ‘immunised’ animal.
An example, to derive a secondary antibody that will recognize a ‘rabbit’ primary antibody,
you could, utilise peptides derived from rabbit IgG FC regions, and immunise a mouse with
these peptides. The mouse would mount an immune response against the peptides
including production of B cell ‘clones’ that produce antibodies that specifically recognize and
bind to epitopes contained within the rabbitIgG FC peptides.
Secondary antibodies are usually labeled (conjugated) with some kind of reporter
system to make them detectable in immunoassays. Detection is usually via a chemical
reaction (e.g. ECL), radiological or more commonly now fluorescent label detection (e.g.
Dylight, Alexa).
Lab work
Day 1 Overview
Day 1 will be a full day lab work.
• In your group of 2 (see Groups below for details), you will first perform a set of immunoprecipitation (IP)
reactions (see Immunoprecipitation IP)
• During the IP stage 2 antigen (epitope) binding incubation, each group will prepare an SDS-PAGE gel
(see SDS-PAGE gel preparation)
• Groups will then load and run their SDS-PAGE gels. Gel runs will take about 45mins -to- 1hr to complete
• Each group will then then be transferred (blot) the protein from their gel onto a LF-PVDF membrane
• The membranes will then be ‘blocked’ and then ‘probed’, overnight, with
a solution containing primary antibody (see antibodies)
Day 2 Overview
Day 2 should be about half a day lab work followed by results overview and discussion
• First each group will wash their membrane and then probe them with a secondary antibody solution
• Membranes will then be washed again and finally anti-TROVE2 antibody signals will be detected using the
BioRadChemiDocMP gel documentation system and ImageLab software. This will be the finish of the lab
work
• We will then discuss results from each group
Protocols
Immunoprecipitation (IP)
Stage 1: Antibody Binding
1) Take 2 x 1.5 ml tubes: label one B (for beads only control) label the other P (protein sample)
2) Thoroughly resuspend the SureBeads solution (green 1.5mL tube) and transfer 100μl of beads
solution to tubes B and P
3) Washing the beads:
a) Magnetise the beads and discard the supernatant
b) Add 1,000μl PBST* wash solution to each tube
c) Remove the tubes from magnet and resuspend the beads thoroughly, by vortexing
d) Flash spin tubes and proceed to back to a)
4) Repeat wash protocol twice more (for a total of 3 washes)
5) Magnetise the beads and discard all wash buffer
6) Add 200μL of anti-TROVE2 monoclonal antibody** solution to tube P
7) Add 200μl of PBS to tube B
8) Thoroughly resuspend the beads in each tube, by vortexing
9) Rotate all tubes for a minimum of 10 min, at room temperature (RT).
* PBST Wash Buffer: PBS + Tween 20 at 0.1% final concentration
**1μg/100μl – antibody/PBS
Stage 2: Antigen (epitope) Binding
10) Wash the beads:
a) Magnetise beads and discard supernatant
b) Add 1,000μl PBST wash solution to each tube of beads
c) Remove tubes from magnet and resuspend the beads thoroughly, by vortexing
d) Flash spin tubes
11) Repeat wash protocol twice more (for a total of 3 washes)
12) Magnetise beads, discard all wash buffer
13) Add 200μl of protein extract (containing 1μg/μl of protein in PBS/RIPA buffer) to both tubes P and B
14) Rotate all tubes for a minimum of 1hr at room temperature (RT)
• Whilst this incubation is underway, each pair of students should prepare a 10% SDS-PAGE
gels See SDS-PAGE gel preparation BioRad FastCastâ„¢ TGX Stain-freeâ„¢ 10%
Stages 3 and 4: Antigen Elution
15) Label 2 x 1.5 ml tubes PD (for protein sample) or BD (for beads only control).
16) Magnetize the beads in tubes P and B and transfer the solutions to the appropriate tube (P to PD, B
to BD). These tubes will contain the TROVE2 IP ‘depleted’ protein extracts
17) Wash the beads in tubes P and B
a) Magnetise beads and discard the supernatant
b) Add 1,000μl PBST wash solution to each tube of beads
c) Remove tubes from magnet and resuspend the beads thoroughly, by vortexing
18) Flash spin tubes, re-magnetize beads, discard supernatant and add 1,000μl PBS-T*
19) Repeat wash protocol once more (for a total of 2 washes)
MSE-4041 Practical 2017-18
3
20) Final wash:
a) Add 1,000 μl PBS-T to each tube, resuspend the beads and transfer to 2 clean 1.5 ml tubes: P1 (for
protein), B1 (for beads only control)
b) Magnetise beads and discard all supernatant
c) Remove beads from magnet and resuspend thoroughly, by vortexing.
21) Spin down both tubes for 30 seconds at 11,000 rpm
22) Magnetize the beads and aspirate off any residual wash buffer from the tubes.
23) Remove beads from magnet, add 40μl 1x Laemmli reducing buffer and incubate for 10 min at 70°C
24) Magnetize beads and transfer the buffer solution (eluent) to 2 clean 1.5 ml tubes labelled: PE (for
protein), or BE (for beads only). Store tubes at 4°C until ready to load the gel
SDS-PAGE gel preparation
Watch the video at this link casting the SDS-PAGE gel to see how to make your gel. Note after adding the TEMED
the gel will start to polymerize, so do not add this reagent until you are ready to pipette the gel mixture between
the casting plates.
One set of BioRad MiniProtean SDS-PAGE gel apparatus: glass plates and casting stand
BioRad FastCastâ„¢ TGX Stain-freeâ„¢ acrylamide solutions
• 1 x tube labelled RA – Resolver A buffer (3mL)
• 1 x tube labelled RB – Resolver B buffer (3mL)
• 1 x tube labelled SA – Stacker A buffer (1mL)
• 1 x tube labelled SB – Stacker B buffer (1mL)
1 x 1.5mL red tube containing TEMED solution (10μL)
1 x 1.5mL green tube containing 10% (w/v) ammonium persulfate solution (50μL)
1. Make 1L of x1 Tris/Glycine SDS (TGS) buffer. This will be sufficient to run 4 gels in one tank.
2. Assemble your gels into a gel tank. Watch the video to see how to assemble
BioRad MiniProtean SDS PAGE gel apparatus.
3. Fill the inside of the running stand with running buffer until it overflows into the tank and carry on filling
until the minimum running level is reached (marked on the side of the gel tank).
SDS-PAGE gel loading and running
Load the protein markers and protein samples into the wells of your gel in the following order:
Run gels at 150v, until the blue loading dye has just run off the bottom of the gel.
Once your gel has run…
• Place about 10mL of ‘used’ running buffer into a plastic tray provided.
• Carefully disassemble the glass plates from the holder and ‘float’ your gel from the glass plate into the
running buffer in the tray (a demonstrator will show you how to do this).
• Next bring your gel-tray to demonstrator and activate the StainFree dye and image the proteins in
the gel using the GelDocEZ imaging system and ImageLab software (see Gel imaging and StainFree dye activation).
• After imaging, transfer the gel back into the tray containing the running buffer and proceed to TBT
electro transfer (see Electrotransfer of protein to LF-PVDF membranes)
Electrotransfer of protein to LF-PVDF membranes.
Watch this video for an overview of the TBT transfer process (you will not be using the premade stacks).
It is essential to minimise none-specific background. Ensure all buffers and reagents have been filtered prior to
use and ‘minimise’ potential dust/debris contact with the membrane surface.
• Remove the lid from a blotting tray, place in it a bottom filter paper/TBT transfer stack, and add
sufficient TBT transfer buffer to fully wet the filter papers
• Place a membrane onto the top of filter paper stack and full wet with TBT transfer buffer
• Place the gel onto the membrane
• Take another set of TBT filter papers, moisten with TBT transfer buffer, and place on top of gel
• Use a roller to gently ‘roll’ over the top layer of filter papers to remove any air bubbles between the filter
papers and the gel.
• Decant ‘excess’ TBT buffer from the lid and place the transfer stack into a TBT transfer cassette.
• Replace the top of the TBT cassette and lock the cassette into place
• Transfer the proteins using the desired settings on the Trans-blot Turbo Transfer System. Watch
this Youtube video for an overview of the TBT system. Although we will not be using the transfer packs the video is
useful for as an overview of the system and transfer.
• We will transfer using BioRad defined ‘mixed molecular weight’ settings (7 minutes – 25mA)
• Checking protein transfer efficiency
• After transfer remove the membrane from the cassette and replace into the tray containing ultrapure
filtered ddH2O
• Pour approximately 10mL of the ultrapure filtered ddH2O onto the surface of the imaging tray and then
place the membrane onto the tray. Make sure the side that was in contact with the gel is side facing upwards
• Image the membrane using the StainFree membrane protocol below.
• Remove the membrane from the imager and replace into the tray containing the ultrapure filtered
ddH2O. Make sure the side of the membrane that was in direct contact with the gel is facing upwards.
• Proceed to western blotting
Western Blot
It is essential to minimise none-specific background. Ensure all buffers and reagents have been filtered prior to
use and ‘minimise’ potential dust/debris contact with the membrane surface.
BioRad TBT LF-PVDF membrane and transfer stack
Blocking solution: x1 MitoSciences blocking solution, diluted in 1 x Phosphate buffered saline (PBS)
TBST Wash Buffer: 20mM Tris, 500mM NaCl (TBS), Tween 20 at 0.05% final concentration in ultra pure filtered
water
Antibodies:
• WB Primary: ab207416 (1/1000 dilution) overnight 4°C
• WB Secondary: SA510034 Goat anti-Rabbit IgG (H&L chains) polyclonal 650 DyLight® pre-adsorbed 1/2000
Day 1
1. Place your membrane section, protein side up (the side of the membrane that was in direct contact
with the gel), into a clean flat plastic tray containing 10mL of x1 blocking solution (1) and incubate for 30
minutes at 4°C
2. Decant blocking buffer and add 10mL of primary antibody solution to the membranes
3. Membranes will then be incubated overnight (16hrs minimum) at 4°C, on a rocking platform
Day 2
1. After incubation, decant and save used primary antibody solution(s)
2. Add 10mL of x 1 wash buffer* and incubate for 5 minutes at room temp
3. Decant used wash solution, add 10mL of x 1 wash buffer and incubate for 5 minutes at room temp
4. Decant used solution, add 10mL of x 1 wash buffer and incubate for 5 minutes at room temp
5. Add 10mL of x1 secondary antibody(s) solution and incubate for 90mins at 4°C
6. Decant used secondary antibody solution, add 10mL of x 1 wash buffer and incubate for 5 minutes at
room temp.
7. Decant used wash solution, add 10mL of x 1 wash buffer and incubate for 5 minutes at room temp.
8. Decant used wash solution, add 10mL of x 1 wash buffer and incubate for 5 minutes at room temp.
9. Decant used wash solution, add 10mL of PBS solution.
10. After washing pour approximately 2mL of the ultrapure filtered ddH2O onto the surface of
the ChemiDocMP imaging tray, then place the membrane onto the imaging tray gel contact side facing
up. Ensure there are no ‘bubbles’ and then image the membrane using the ChemiDocMP
11. Unless stated, all incubations are carried out at room temperature on a ‘rocking’ shaker set at 1
rotation/second.
Membrane Imaging using the BioRad ChemiDocMP
Turn the imager on. Click the 3 channel intense band.mptl link below to start ImageLab. Then load the imaging
protocol. Wait for the camera to cool down to operating temperature.
Initial imaging settings
Red channel: – Dylight 650 autoexposure, intense bands
Green channel: Dylight 549 – autoexposure, intense bands
Blue channel: StainFree autoexposure, intense bands
Practical: Assessment report write up
guide and marking criteria
Assessment breakdown
The report should be formatted as if it were to be submitted to the Journal Cell. The
highlights/abstract, Introduction, Results and Discussion sections will be marked.
Marks breakdown
•
•
•
•
Highlights/in brief/Summary (10%)
Introduction (30%)
Results (30%)
Discussion (30%)
Highlights/Summary
Highlights 3-4 highlights – focused on the report
Summary Approximately 150 words (±10%)
Introduction
Approximately 500 words
The introduction must cover what has been previously published in the literature about the
research topic. It should also give background information on the experimental procedures
being used and a brief overview of the overall experimental plan. It should not contain any
actual results from the experiment to be performed. Introductions MUST be sufficiently and
correctly referenced with relevant primary literature/reviews from peer reviewed scientific
journals.
This report introduction must ‘introduce’ at least 3 main areas
1. The previous applications of immunoassays in research
2. Immunoprecipiation and western blot detection of proteins
3. Focused overview of the aims of the practical
Results
Approximately 500 words (excluding figure/tables legends)
See Practical: Results and feedback for data files
The results should contain a number of sections, each presenting an explanatory text and fully
processed data/results figures/tables related to a specific aim or set of aims and objectives.
You must decide how many ‘results sections’ should be presented and how best to structure
and order these sections to enable a logical progression through the experiments performed,
to a set of ‘final results/conclusions’ achieved.
Each result section must contain
1. A main text presenting fully processed results/data for the particular result section
2. Figure(s) and/or tables/graphs presenting fully processed data
Figures for this report. This section MUST contain the following fully
processed results figures
• Figure 1 A fully processed and annotated multi-channel (650, 549 and StainFree)
western blot image of your results
• Figure 2 A fully processed and annotated multi-channel western blot image (650, 549
and StainFree) of the ‘most interesting*’ data set from all the class western
blot membranes
*This will be determined in the discussion feedback session
NOTE: All figures and/or tables must support the results presented in the text and be cross
referenced from the text. Both figures must be presented on a single page, NOT ‘fitted within’
the text sections of the results. Each figure must have a suitable title and legend (below the
figure); which must provide note form information on any key data/points in the figure. The
legend should enable the figure to ‘stand alone’ i.e. the reader should not have to refer back to
the main text for key information on how to interpret or understand the main findings
presented in the figure. Notes on figure data image ‘cropping’. Generally, if an immunoblot
image is cropped it MUST be in a way that retains information about antigen size and
antibody specificity. The cropped images should retain sufficient area around the band(s) of
interest, ideally including the positions of at least one molecular weight marker above and
below the band(s).
Discussion
Approximately 800 words (excluding any figures, tables and legends)
The discussion must be correctly and well referenced with current relevant literature from
peer reviewed scientific sources. The discussion should have 3 sections.
• Section 1: Clearly discuss the main findings from each section and how one results
section leads on to the next. Highlight any issues that may have affected the
accuracy/precision of your results and/or progression from one stage to the next stage.
This section should be a logical progression of the ‘flow’ of the whole
experiment/practical from start to finish.
• Section 2: You should then come to a final conclusion and discussion point as to the
success or otherwise of the entire experiment(s). Discuss your results in comparison to
what’s been previously published in the literature. What conclusions can you draw from
your data and the final result and what are the key impact(s) of your results? Where
possible, these should be compared to previous published findings.
• Section 3: Finally, discuss why should other people care about this ‘result’ and what
could you or they do to further advance this work?
Suitability for publication in a peer reviewed scientific journal
Note for PLSP: This criteria will be applied in the context of the specific
applicable PLSP guidelines
In addition to the knowledge and critical thinking understanding this assessment will gauge
the ‘presentation’ quality of report as a whole. It will judge the quality of and how clear and
easily interpreted are the figures and tables, the level of concise Scientific English used and
the quality/quantity of referencing in the text and final bibliography.
1. Presentation quality of the data:
o All figures must be presented as outlined in the guidelines and the template
document. All figures must be fully processed with key all data clear and
unobscured. Figures must be fully annotated (e.g. arrows, lane numbers,
sample identification, etc. Annotations must not obscure any figure data.
2. Scientific English:
o You’re writing this thesis/report for scientists, who expect to read
accurate unambiguous scientific English. Sentences like ‘the band is bright’ or ‘I
saw a fat band in the gel’ are examples of poor scientific English. Band 1 shows a
higher intensity relative to band 2, indicating it contains a higher
concentration of DNA’ is an example of higher quality scientific English. If, for
example, you can attach figures to the intensity, and refer to these either in a table
or the main text, even better.
3. Quality/quantity of referencing and final bibliography
o For research to pass ‘peer review’ and be published in a quality journal, the paper
presenting it must contain sufficient current relevant background literature to
support the experimental plan, and set the findings and final discussion/conclusions
into context. Without such referencing you will not be able to publish your
research in a quality journal.

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