Bridges Lab Protocols - User contributions [en]

Immunofluoresence

2012-07-11T22:35:59Z

Irith:

==Reagents==
*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for lipid/membrane bound proteins) in PBS or ice cold 10% methanol (for cytoskeleton bound proteins)
*Cold PBS
*100 mM Glycine in PBS
*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
*Blocking Solution: 2% BSA PBS
*Vectashield

==Protocol==
#Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
#Treat cells as required
#Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
#Wash twice with PBS
#Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
#Wash once with PBS
#Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
#Wash three times with PBS (5 min each)
#Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
#Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
#Wash coverslips 3 times 5 minutes with PBS with gentle rocking
#Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
#Wash coverslips 3 times 10 minutes with PBS with gentle rocking
#Add 10 uL vectashield to glass slide and place cells on slide (coverslip side up, use sharp tweezers and tip, 2 coverslips on each slide). Label slide with date, cells and treatment + color code for antibodies. Fix with nail polish, dry for at least 1 hour up to ON in dark drawer. Store long term in sealed box at 4C.

*It is possible to use ImageJ to analyse [[Colocalization]]
[[Category:Immunofluoresence]]

Glucose Uptake Assay

2012-07-11T21:03:35Z

Irith:

==Materials==
*2-Deoxyglucose (cold): Sigma D-6134. Dissolve 32.8mg/mL (200 mM) in [[KRBH Buffer]]
*0.5% BSA in [[KRBH Buffer]]
*Radioactive <sup>14</sup>C-2-deoxyglucose
*Hot 2-DG. Need 50 uL per well, prepare 1 mL KRBH/BSA, 1 uL cold 2-DG and 50 uL radioactive 2-DG. count 3X5 ul of hot 2DG.

==Protocol==
#Starve cells >3h in 0.5% FBS
#Prepare insulin in KRBH/BSA (0.6uL (100nM) insulin/mL, needing 0.5 mL per well)
#Wash cells 2x with warm PBS -/-
#Add 450 uL KRBH/BSA with or without insulin to wells. Typically do triplicate measurements
#Wait 30 min
#Add 50 uL hot 2-DG solution per well and start timer
#After 5 min add 50 uL cold 2-DG
#Wash cells 3x1mL with cold PBS
#Add 500 uL PBS per well
#Scrape cells with an upside down p200 tip
#Add 400 uL cells to 5 mL aqueous scintillation fluid, vortex and count on program 3
#Do a bradford assay on 50 uL of cells (use PBS as blank)

==Analysis==
*For excel fill in orange cells in this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/Excel%20Templates/2-DG%20Template.xlsx]
*For sweave use this template found on Github [https://github.com/davebridges/biomolecule-scripts/blob/master/R/Sweave/2-dg%20analysis.Rnw]

[[Category: Metabolic Measurements]]
[[Category: Glucose Uptake]]
[[Category: Cell Culture]]

Triglyceride Assay from Cells and Tissues

2012-07-11T21:01:45Z

Irith:

==Materials==
* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat 337-B)

==Protocol==
# Weigh out 200-500mg tissue (record weight for normalization). You can use as low as 30 mg tissue if necessary by reducing the lysis volume (must be at least 500 uL) or increasing the amount of the lower (chloroform) layer removed up to 500 ul(normally 180 uL).
# Homogenize with dounce homogenizer in 2 mL '''Homogenization Buffer'''.
# Remove 200 uL to a tube containing 5 uL KOH
# Mix by inverting
# Add 800 uL '''Chloroform/Methanol Mixture'''
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take 200 uL of the bottom layer into a fresh tube. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
## Resuspend triglyceride and glycerol reagent with water if necessary.
## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.

===Suggested Volumes===
This is based on using a 96 well plate to measure final concentrations.
{| border="1"
|-
! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume
|-
| Liver || 1 mL || 200 uL || 500 uL || 5 uL
|-
| Muscle || 1 mL || 200 uL || 50 uL || 5 uL
|}

Lipofectamine Plasmid Transfection

2012-07-11T21:00:28Z

Irith:

[[Category: Cell Culture ]]
[[Category: Transfection]]

==Materials==
*Cells in a 6 well dish, plated and at >90% confluence
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)

==Required Amounts==
*Calculate DNA to transfect
*Lipofectamine(in uL) = DNA(in ug) * 2.5
*OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 25

==Protocol (6 well dish)==
#Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG).
#For each well prepare:
##250uL OptiMEM plus 4 ug of DNA.
##250uL OptiMEM plus 10 uL of Lipofectamine.
##Incubate ~5 minutes at room temperature.
##Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine.
##Incubate ~20 min at room temperature.
#Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells
#Gently rock plate to mix
#After ~4h aspirate media and re-feed cells with normal media. (It is usually possible to leave overnight).

Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual]

Generating and Amplifying Adenoviral Constructs

2012-07-11T20:56:45Z

Irith:

__NOTOC__
[[Category:shRNA]][[Category:Adenovirus]][[Category:Transduction]][[Category:Knockdown]][[Category:Molecular Biology]]

==Materials==
*Targetting Construct in pAdtrack vector or the like (see [[ Preparing an Adenoviral shRNA Clone ]])
*PmeI restriction enzyme (or other enzyme as required for linearization.
*PacI restriction enzyme
*Electrocompetent BJ5183-AD-1 cells (Agilent Cat#200157; or prepare yourself)
*7.5 M ammonium acetate
*20mg/mL glycogen
*25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0
*100% ethanol
*2 mm electroporation cuvette
*25 cm2 and 75 cm2 tissue culture flasks.
*Ultraclear pollyallomer tubes for Ti45 rotor (Beckman Cat# 344087)
*2x Virus storage buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA and 50% glycerol; filter sterilized)

==Protocol==
see [http://www.coloncancer.org/adeasy/He,%20AdEasy%20-%20Nature%20Protocols,%202007.pdf Nature Protocols], [http://onlinelibrary.wiley.com/doi/10.1002/0471142905.hg1204s40/pdf Current Protocols in Human Genetics] protocols.
===Linearization and Purification of Shuttle DNA===
#Digest 0.2-0.5 ug plasmid DNA in 100 uL volume with 30-100U of PmeI overnight at 37C. If desired check a small amount by electrophoresis to ensure complete ligation.
#Add 100 uL ddH2O, 100 uL 7.5 M ammonium acetate and 2 uL 20 mg/ml glycogen.
#Add 300 uL 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0.
#Remove top layer to clean tube and add 600 uL of 100% ethanol
#Centrifuge 5 min at 13 000 RPM
#Wash three times with 70% ethanol to remove residual salt.
#Dry and resuspend in 8 uL

===Transformation into BJ5183-AD-1 cells===
#Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
#Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
#Electroporate at '''2,500 V, 200 O and 25 mF'''
#Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
#Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
#Miniprep clones. Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel. Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
#Retransform and amplify correct clone(s).

===Transformation into 293A Cells===
#Plate cells so that at time of transformation they are 50-70% confluent.
#Digest recombinant plasmid with PacI in 100 uL with 3ug plasmid and 100U PacI.
#Precipitate with 70% ethanol, dry and resuspend in 20 uL sterile water.
#Mix 3 ug PacI digested plasmid and 15 uL LipofectAMINE reagent for each 25-cm2 tissue culture flask in 500 uL Opti-MEM I, and incubate the DNA/LipofectAMINE reagent mix for 15–30 min at room temperature.
#While waiting, wash cells with serum free DMEM and add OptiMEM (5 mL/10 cm dish)
#Add DNA/Lipofectamine and incubate 4-6h.
#Replace media with complete DMEM
#The next day check transformation efficiency by GFP fluoresence (for pAdtrack)
#Do not remove media, but add 2 mL fresh complete DMEM every 5-7 days. Wait 2-3 weeks before collecting viral particles.

===Preparation and Purification of Viral Particles===
#Scrape cells into 15 mL conical tube.
#Aspirate all but the final 2 mL of cells and resuspend by vortexing
#Release viral particles with four freeze-thaw cycles (liquid nitrogen then 37C water bath)
#Centrifuge at 500g at 4C to pellet debris.
#Store supernatant (virus) at -80 or use immediately to amplify

===Amplification of Adenovirus===
#Plate 293A cells in 25-cm2 tissue culture flasks at 80–90% confluency in 7 ml complete DMEM 6–15 h before infection.
#Infect 293A cells by adding 40–50% of the primary transfection viral supernatants (i.e., 0.5–1.0 ml of the 2.0 ml viral lysate) to each 25-cm2 flask.
#Check transduction by GFP fluorsesence (for pAdtrack)
#Collect cells by scraping into a 15 mL conical 3-5 days post-infection.
#Remove all but 5 mL media by aspiration.
#Freeze/Thaw cells 4 times as described above
#Centrifuge at 500g to pellet debris
#Store supernatant (virus) at -80 or use immediately to amplify again.
#Repeat amplification three time more first with one 75 cm2 flask (using entire supernatant) then with 3-5 75 cm2 flasks, then with 10-20 75cm2 flasks using entire viral supernatant as the innoculant each time.
#For final amplification resuspend pelleted cells in 8 mL D-PBS -/-, perform 4 freeze thaw cycles and centrifuge 10 min at 7000g

===Purification of Adenovirus===
#Transfer supernatant to 50 mL conical tube and add 4.8g of Cesium chloride and dissolve
#Transfer 10 mL to 12 mL polyallomer tubes (for SW 45-Ti) add ~ 2mL mineral oil to top of tube to prevent tube crushing.
#Centrifuge 18–24 h at 176,000g (SW 41 Ti rotor at 32,000 r.p.m.)
#The purified virus should be 1-2cm below the mineral oil in an opaque layer.
#Remove virus layer to a clean falcon tube using a syringe (do this in a beaker to easier add bleach to waste).
#Add an equal volume of 2X Virus storage buffer and store at -80
#Titer the virus using a kit.

===desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)===
# Cut column, discard storage solution and wash X5 with ~5ml PBS
# Add virus sample (up to 2.5 ml)
# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
# dilute to 1 OD/ml
# Add 10% glycerol and store at -80.

===Tail Vein Injection===
# Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.
# Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).
# Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.

Triglyceride Assay from Cells and Tissues

2012-07-11T20:52:18Z

Irith:

==Materials==
* '''Homogenization Buffer''' (50 mM Tris, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat 337-B)

==Protocol==
# Weigh out 200-500mg tissue (record weight for normalization). You can use as low as 30 mg tissue if necessary by reducing the lysis volume (must be at least 500 uL) or increasing the amount of the lower (chloroform) layer removed up to 500 ul(normally 180 uL).
# Homogenize with dounce homogenizer in 2 mL '''Homogenization Buffer'''.
# Remove 200 uL to a tube containing 5 uL KOH
# Mix by inverting
# Add 800 uL '''Chloroform/Methanol Mixture'''
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take 200 uL of the bottom layer into a fresh tube. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
## Resuspend triglyceride and glycerol reagent with water if necessary.
## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.

===Suggested Volumes===
This is based on using a 96 well plate to measure final concentrations.
{| border="1"
|-
! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume
|-
| Liver || 1 mL || 200 uL || 500 uL || 5 uL
|-
| Muscle || 1 mL || 200 uL || 50 uL || 5 uL
|}

Immunoprecipitation

2012-07-03T19:33:40Z

Irith:

==Protocol==

Samples are always kept on ice/in cold room/4 celsius centrifuge
* wash cells once with PBS
* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors
* for each well in a 12 well plate lyse in 1 ml.
* combine 0.5 ml lysate, 0.2-0.5 ug of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
* for tissue lysates - use 0.5 mg of protein, add primary antibody first for 0.5 hour and then add beads.
* rotate 1 hour to overnight in cold room
* spin 2 minutes at 5000 rpm

and aspirate supernatent, carefully avoiding beads (can use crushed tip).
* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.
* after last wash and aspiration, spin once more and aspirate all supernatant.
* add 50 ul of sample buffer, boil 5 minutes.
* to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder, as in picture [[File:Needle_in_eppendorf_2.jpg|100px]]). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm.
* load 10ul of IP on gel.
* load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer).

Glycogen Determination from Tissues

2012-06-08T14:26:55Z

Irith:

[[Category:Glycogen]]
[[Category:Metabolism]]
[[Category:Mouse Tissues]]
[[Category:Mouse Work]]

==Materials and Buffers==
* Screw Capped Vials
* 30% KOH, prepared fresh
* 1M Sodium Sulfate
* Ethanol
* 50 mM Sodium Acetate, pH 4.8
* Amyloglucosidase 0.3 mg/mL in 50 mM Sodium Acetate. Stored in -80
* Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091; for protocol see http://www.wakodiagnostics.com/pi/pi_autokit_glucose.pdf)
* Glucose standard solution (200 or 500 mg/dL; Wako)

==Protocol==
# Weight out 30-90 mg tissue into a '''screw cap vial''' and record weights. Screw cap vials are really important or else the lids will pop off
# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing.
# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
# Boil for 5 min.
# Centrifuge at 13 000 RPM for 5 min.
# Resuspend pellet in 200 uL water then add 400 uL ethanol. Boil 5 min, spin 5 min and Repeat wash steps twice more.
# Dry pellet on the bench
# Prepare amyloglucosidase solution by diluting the AG stock 100X into 50 mM Sodium Acetate, pH 4.8. Prepare enough for 200 uL per tube plus some extras
# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
# Quantify glucose using kit:
## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
## Add 1-5 uL glucose standard (500mg/dL) for standard curve (for microplate add 1-5 ul of glucose standard 200mg/dL diluted 1:5)
## Add 10 uL digested glycogen (for mice fasted more than 6 hours. for fed/short fast need less)
## Mix and incubate at 37C for 5 min
## Measure absorbance at 505 nm
## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)

==Calculations==
# Use this Sweave template: https://raw.github.com/davebridges/biomolecule-scripts/master/R/Sweave/glycogen-analysis-tissue.Rnw
Reference:

PMID 15282316

Glycogen Determination from Tissues

2012-06-08T14:03:08Z

Irith:

[[Category:Glycogen]]
[[Category:Metabolism]]
[[Category:Mouse Tissues]]
[[Category:Mouse Work]]

==Materials and Buffers==
* Screw Capped Vials
* 30% KOH, prepared fresh
* 1M Sodium Sulfate
* Ethanol
* 50 mM Sodium Acetate, pH 4.8
* Amyloglucosidase 0.3 mg/mL in 50 mM Sodium Acetate. Stored in -80
* Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091; for protocol see http://www.wakodiagnostics.com/pi/pi_autokit_glucose.pdf)
* Glucose standard solution (200 or 500 mg/dL; Wako)

==Protocol==
# Weight out 30-90 mg tissue into a '''screw cap vial''' and record weights. Screw cap vials are really important or else the lids will pop off
# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing.
# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
# Boil for 5 min.
# Centrifuge at 13 000 RPM for 5 min.
# Resuspend pellet in 200 uL water then add 400 uL ethanol. Boil 5 min, spin 5 min and Repeat wash steps twice more.
# Dry pellet on the bench
# Prepare amyloglucosidase solution by diluting the AG stock 100X into 50 mM Sodium Acetate, pH 4.8. Prepare enough for 200 uL per tube plus some extras
# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
# Quantify glucose using kit:
## Add 700 uL Glucose Buffer Solution with Color Reagent to a plastic cuvette. (for microplate add 100ul)
## Add 1-5 uL glucose standard (500mg/dL) for standard curve (for microplate add 1-5 ul of glucose standard 200mg/dL diluted 1:5)
## Add 10 uL digested glycogen
## Mix and incubate at 37C for 5 min
## Measure absorbance at 505 nm
## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)

==Calculations==
# Use this Sweave template: https://raw.github.com/davebridges/biomolecule-scripts/master/R/Sweave/glycogen-analysis-tissue.Rnw
Reference:

PMID 15282316

Amylose pull-down of glycogen-bound proteins

2012-05-07T16:12:15Z

Irith: Created page with 'All steps on ice/cold room Add 50 ul beads (amylose resin #E80021L Biolabs) to 0.5 mg of protein lysate in lysis buffer with protease inhibitors Add 1 ml of lysis buffer Rotate i...'

All steps on ice/cold room
Add 50 ul beads (amylose resin #E80021L Biolabs) to 0.5 mg of protein lysate in lysis buffer with protease inhibitors
Add 1 ml of lysis buffer
Rotate in cold room 30’ to ON
Spin down at max speed for 1’
Aspirate supernatant down to 1 mm over beads with crushed tip.
Repeat 3-5 washes with lysis buffer without protease inhibitors
After last wash and aspiration spin again and aspirate all supernatant
Ad 50ul SDS loading buffer, boil for 5 minutes
Load 10ul/gel

Immunoprecipitation

2012-04-05T15:42:06Z

Irith:

==Protocol==

Samples are always kept on ice/in cold room/4 celsius centrifuge
* wash cells once with PBS
* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors
* for each well in a 12 well plate lyse in 1 ml.
* combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
* rotate 1 hour to overnight in cold room
* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip).
* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.
* after last wash and aspiration, spin once more and aspirate all supernatant.
* add 50 ul of sample buffer, boil 5 minutes.
* to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder, as in picture [[File:Needle_in_eppendorf_2.jpg|100px]]). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm.
* load 10ul of IP on gel.
* load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer).

Immunoprecipitation

2012-04-05T15:29:21Z

Irith:

==Protocol==

samples are on ice/in cold
* Scrape cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see [[RIPA Buffer]]).
* For each well in a 12 well plate lyse in 1 ml.
* combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
* rotate 1 hour-overnight in cold room
* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads
* wash X3: add 1 ml lysis buffer, mix by inverting tube, spin and aspirate supernatent as before.
* spin once more and aspirate all supernatant.
* add 50 ul of sample buffer, boil 5 minutes
* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg|100px]] (up to halfway of bevel).

Immunoprecipitation

2012-04-05T15:29:07Z

Irith:

Immunoprecipitation

2012-04-05T15:28:49Z

Irith:

File:Needle in eppendorf 2.jpg

2012-04-05T15:27:20Z

Irith:

Immunoprecipitation

2012-04-05T15:26:05Z

Irith:

Immunoprecipitation

2012-04-04T16:42:15Z

Irith: Created page with 'Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see link). For each well in a 12 well plate lyse in 1 ml. rotate 0.5 ml lysat...'

Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see link).
For each well in a 12 well plate lyse in 1 ml.
rotate 0.5 ml lysate with 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).

Immunofluoresence

2012-03-23T19:47:27Z

Irith:

==Reagents==
*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for cytoskeleton bound proteins) in PBS or ice cold 10% methanol (for lipid/membrane bound proteins)
*Cold PBS
*100 mM Glycine in PBS
*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
*Blocking Solution: 2% BSA PBS
*Vectashield

==Protocol==
#Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
#Treat cells as required
#Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
#Wash twice with PBS
#Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
#Wash once with PBS
#Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
#Wash three times with PBS (5 min each)
#Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
#Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
#Wash coverslips 3 times 5 minutes with PBS with gentle rocking
#Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
#Wash coverslips 3 times 10 minutes with PBS with gentle rocking
#Add 10 uL vectashield to glass slide and place cells on slide (coverslip side up, use sharp tweezers and tip, 2 coverslips on each slide). Label slide with date, cells and treatment + color code for antibodies. Fix with nail polish, dry for at least 1 hour up to ON in dark drawer. Store long term in sealed box at 4C.

*It is possible to use ImageJ to analyse [[Colocalization]]
[[Category:Immunofluoresence]]

Immunofluoresence

2012-03-22T18:04:23Z

Irith:

==Reagents==
*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for cytoskeleton bound proteins) in PBS or ice cold 10% methanol (for lipid/membrane bound proteins)
*Cold PBS
*100 mM Glycine in PBS
*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
*Blocking Solution: 2% BSA PBS
*Vectashield

==Protocol==
#Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
#Treat cells as required
#Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
#Wash twice with PBS
#Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
#Wash once with PBS
#Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
#Wash three times with PBS (5 min each)
#Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
#Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
#Wash coverslips 3 times 5 minutes with PBS with gentle rocking
#Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
#Wash coverslips 3 times 10 minutes with PBS with gentle rocking
#Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish, dry for at least 1 hour.

*It is possible to use ImageJ to analyse [[Colocalization]]
[[Category:Immunofluoresence]]

Glucose Tolerance Test

2011-08-24T16:19:56Z

Irith:

==Materials (Bring Downstairs)==
*Glucometer - AccuChek Advantage
*Glucose Test Strips - AccuChek Comfort Curve. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [[https://www31.med.umich.edu/materielsvcs/catalog/details.cfm?stock=2535 here]]. collect in university hospital building, floor B2 in room B2F408 dock 5 across from central pharmacy.
*Scale
*Beaker for weighing mice
*Syringes
*10% Glucose in PBS (make as 1g in 10 mL, sterile filtered). This will correspond to 1g/kg glucose injections.
*Timer

==Protocol==
#Remove food from mice for about 6h. Add do not feed tag to cages.
#Bring mice to procedure room. Put water bottle on cage.
#Weigh mice, mark tails as necessary and take fasting glucose measurement. If necessary also take blood for [[Measuring Fasting Insulin]] by filling a heparinized capilary about 2/3 full with blood.
#Prepare glucose syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).
#At 1 min intervals, inject appropriate amount of glucose into interperitoneal cavity of the mouse.
##Immobilize mouse and restrain tail with one hand
##Aim needle between the midline and the hip bone
##Insert syringe (do not inject) into cavity
##Pull up syringe and ensure that only air is aspirated. If urine or blood appears in the syringe, start over
##Eject syringe.
#At desired intervals (normally 15, 30, 45, 60, 75, 90 and 120 min), take blood glucose measurements from tail vein

[[Category:Mouse Work]]
[[Category:Glucose Homeostasis]]
[[Category:Injections]]

Talk:Glucose Tolerance Test

2011-08-22T20:23:27Z

Irith: Created page with 'for Shannon's glucometer strips https://www31.med.umich.edu/materielsvcs/catalog/details.cfm?stock=25704'

for Shannon's glucometer strips https://www31.med.umich.edu/materielsvcs/catalog/details.cfm?stock=25704

Generating and Amplifying Adenoviral Constructs

2011-03-23T19:18:27Z

Irith: /* Purification of Adenovirus */

__NOTOC__
[[Category:shRNA]][[Category:Adenovirus]][[Category:Transduction]][[Category:Knockdown]][[Category:Molecular Biology]]

==Materials==
*Targetting Construct in pAdtrack vector or the like (see [[ Preparing an Adenoviral shRNA Clone ]])
*PmeI restriction enzyme (or other enzyme as required for linearization.
*PacI restriction enzyme
*Electrocompetent BJ5183-AD-1 cells (Agilent Cat#200157; or prepare yourself)
*7.5 M ammonium acetate
*20mg/mL glycogen
*25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0
*100% ethanol
*2 mm electroporation cuvette
*25 cm2 and 75 cm2 tissue culture flasks.
*Ultraclear pollyallomer tubes for Ti45 rotor (Beckman Cat# 344087)
*2x Virus storage buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA and 50% glycerol; filter sterilized)

==Protocol==
see [http://www.coloncancer.org/adeasy/He,%20AdEasy%20-%20Nature%20Protocols,%202007.pdf Nature Protocols], [http://onlinelibrary.wiley.com/doi/10.1002/0471142905.hg1204s40/pdf Current Protocols in Human Genetics] protocols.
===Linearization and Purification of Shuttle DNA===
#Digest 0.2-0.5 ug plasmid DNA in 100 uL volume with 30-100U of PmeI overnight at 37C. If desired check a small amount by electrophoresis to ensure complete ligation.
#Add 100 uL ddH2O, 100 uL 7.5 M ammonium acetate and 2 uL 20 mg/ml glycogen.
#Add 300 uL 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0.
#Remove top layer to clean tube and add 600 uL of 100% ethanol
#Centrifuge 5 min at 13 000 RPM
#Wash three times with 70% ethanol to remove residual salt.
#Dry and resuspend in 8 uL

===Transformation into BJ5183-AD-1 cells===
#Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
#Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
#Electroporate at '''2,500 V, 200 O and 25 mF'''
#Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
#Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
#Miniprep clones. Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel. Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
#Retransform and amplify correct clone(s).

===Transformation into 293A Cells===
#Plate cells so that at time of transformation they are 50-70% confluent.
#Digest recombinant plasmid with PacI in 100 uL with 3ug plasmid and 100U PacI.
#Precipitate with 70% ethanol, dry and resuspend in 20 uL sterile water.
#Mix 3 ug PacI digested plasmid and 15 uL LipofectAMINE reagent for each 25-cm2 tissue culture flask in 500 uL Opti-MEM I, and incubate the DNA/LipofectAMINE reagent mix for 15–30 min at room temperature.
#While waiting, wash cells with serum free DMEM and add OptiMEM (5 mL/10 cm dish)
#Add DNA/Lipofectamine and incubate 4-6h.
#Replace media with complete DMEM
#The next day check transformation efficiency by GFP fluoresence (for pAdtrack)
#Do not remove media, but add 2 mL fresh complete DMEM every 5-7 days. Wait 2-3 weeks before collecting viral particles.

===Preparation and Purification of Viral Particles===
#Scrape cells into 15 mL conical tube.
#Aspirate all but the final 2 mL of cells and resuspend by vortexing
#Release viral particles with four freeze-thaw cycles (liquid nitrogen then 37C water bath)
#Centrifuge at 500g at 4C to pellet debris.
#Store supernatant (virus) at -80 or use immediately to amplify

===Amplification of Adenovirus===
#Plate 293A cells in 25-cm2 tissue culture flasks at 80–90% confluency in 7 ml complete DMEM 6–15 h before infection.
#Infect 293A cells by adding 40–50% of the primary transfection viral supernatants (i.e., 0.5–1.0 ml of the 2.0 ml viral lysate) to each 25-cm2 flask.
#Check transduction by GFP fluorsesence (for pAdtrack)
#Collect cells by scraping into a 15 mL conical 3-5 days post-infection.
#Remove all but 5 mL media by aspiration.
#Freeze/Thaw cells 4 times as described above
#Centrifuge at 500g to pellet debris
#Store supernatant (virus) at -80 or use immediately to amplify again.
#Repeat amplification three time more first with one 75 cm2 flask (using entire supernatant) then with 3-5 75 cm2 flasks, then with 10-20 75cm2 flasks using entire viral supernatant as the innoculant each time.
#For final amplification resuspend pelleted cells in 8 mL D-PBS -/-, perform 4 freeze thaw cycles and centrifuge 10 min at 7000g

===Purification of Adenovirus===
#Transfer supernatant to 50 mL conical tube and add 4.8g of Cesium chloride and dissolve
#Transfer 10 mL to 12 mL polyallomer tubes (for SW 45-Ti) add ~ 2mL mineral oil to top of tube to prevent tube crushing.
#Centrifuge 18–24 h at 176,000g (SW 41 Ti rotor at 32,000 r.p.m.)
#The purified virus should be 1-2cm below the mineral oil in an opaque layer.
#Remove virus layer to a clean falcon tube using a syringe (do this in a beaker to easier add bleach to waste).
#Add an equal volume of 2X Virus storage buffer and store at -80
#Titer the virus using a kit.

===desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)===
# Cut column, discard storage solution and wash X5 with ~5ml PBS
# Add virus sample (up to 2.5 ml)
# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
# Add 10% glycerol and store at -80.

===Tail Vein Injection===
# Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.
# Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).
# Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.