Antibodies
Antibodies kdorfman Tue, 10/02/2018 - 16:21For initial dilution of lyophilized antibody:
Dissolve to recommended concentration (per package insert) with half glycerol and half water.
Aliquot and freeze.
Label the tubes or the box with how much to dilute.
Primary Antibodies
Primary Antibodies kdorfman Tue, 10/02/2018 - 16:215-mC (rabbit)
5-mC (rabbit) kdorfman Fri, 11/05/2021 - 13:445-methylcytosine (D3S2Z)
Rabbit mAb
Binds to methylated cytosine
28692S Cell Signaling Technology
From the manufacturer: "Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1
Dilute 1:1600
Before Antibody treatment, treat fixed cells with 4N HCl for 15 minutes to denature DNA.
Neutralize with 2 washes with sodium borate pH 9, then PBS. Then start the PBS-Tw-Az rinses before incubating with the antibody.
Store at -20
Actin (mouse)
Actin (mouse) kdorfman Tue, 10/02/2018 - 18:50stock concentration = 22 ug/mL
working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 90 uL ~11 mg/mL
Add 216 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Actin (rabbit)
Actin (rabbit) kdorfman Tue, 10/02/2018 - 19:04Dilute 1:40 for use
Dilute 1:1 with glycerol
Aliquot 16 uL
Add 290 uL PBS BSA to make enough for 3 slides
Freezer 266A
Alkaline phosphatase (mouse)
Alkaline phosphatase (mouse) kdorfman Tue, 10/02/2018 - 18:51stock concentration = 38 ug/mL
diluted 1:1 in glycerol
50 uL aliquots of ~19ug/mL
Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides
266A freezer
Alpha-actinin (mouse)
Alpha-actinin (mouse) kdorfman Tue, 10/02/2018 - 17:46Binds actin to membrane at adherin junctions
Mouse monoclonal, Clone BM 75.2
IgM
Fix in formaldehyde (no MeOH or glutaraldehyde)
1:200
Use anti-mouse IgM FITC secondary
Cadherin (mouse)
Cadherin (mouse) kdorfman Tue, 10/02/2018 - 17:37"calcium-dependent adhesion"
cell adhesion molecule (CAM) important in formation of adherens junctions to bind cells with each other.
Class of type-1 transmembrane proteins.
Dependent on calcium (Ca2+) ions to function
small aliquot: 5 µL
large aliquot: 10 µL
Collagen II
Collagen II kdorfman Tue, 10/02/2018 - 18:59stock solution: 96 ug/mL
working solution: 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 20 uL ~48 mg/mL
Add 286 uL PBS-Tw-BSA to make enough for 3 slides
266A Freezer
Collagen IV
Collagen IV kdorfman Tue, 10/02/2018 - 19:00mouse anti collagen IV (basement membranes)
2 tubes as of spring 2018:
- stock concentration (8/7/08) = 30 ug/mL
- stock concentration (12/22/11) = 39 ug/mL
working concentration = 2 - 5 ug/mL
Each diluted with glycerol to a concentration of 18.5 ug/mL
Aliquots are 50 uL
Add 256 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Connexin (rabbit)
Connexin (rabbit) kdorfman Tue, 10/02/2018 - 19:07Freezer 266A
Anti-connexin-32 (265-279)
Dilute 1:600
Anti-Connexin-32 (106-124)
Dilute 1:400
Connexin (rat)
Connexin (rat) kdorfman Tue, 10/02/2018 - 19:17stock concentration = 28 ug/mL
working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 70 uL ~14 mg/mL
Add 236 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
DNMT1 (rabbit)
DNMT1 (rabbit) kdorfman Fri, 11/05/2021 - 13:39D63A6 XP(R)
Rabbit mAb
5032S Cell Signalling Technology
detects endogenous levels of total DNMT1 protein.
From the manufacturer: "Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication."
Dilute 1:100 for use
Golgi (mouse)
Golgi (mouse) kdorfman Tue, 10/02/2018 - 17:111 µL aliquots in tube. Store in freezer (-20C)
add 100 µL 1% BSA in PBS
Use just under 50 µL per coverslip (so there is enough for 2 per tube)
[edited 9/26/18 - used to say 1:200 dilution, but we had better results with 1:100]
Hec1 (mouse)
Hec1 (mouse) kdorfman Tue, 10/02/2018 - 16:23Novus Biologicals, recombinant human Hec1, monocolonal
fix in methanol
dilute 1:200
Histone H3 phospho S10
Histone H3 phospho S10 kdorfman Fri, 11/04/2022 - 20:46FINISH WORKING ON THIS!
Rabbit polyclonal
1 mg/mL, 100 ug, should be 100 uL in the package
1:2000
Storage:
- Dilute 1;10 glycerol
- 2 uL aliquots; for use, raise to 400 uL
- leave the rest with a label: dilute 1 uL in 200 uL PBS-Tw-Az-BSA
Integrin (rat)
Integrin (rat) kdorfman Tue, 10/02/2018 - 19:18rat anti-integrin-3
IgG2a
stock concentration = 27 ug/mL
working concentration = 2 - 5 ug/mL
Freezer 266A
LAMP1 (lysosomes) (mouse)
LAMP1 (lysosomes) (mouse) kdorfman Tue, 10/02/2018 - 16:22IgG
1 mg/mL
4C short term
Freeze 1 µL aliquots
dilute 1:100 - 1:750 in 0.1% BSA in PBS
rinse in PBS
Fix
- in 4% paraformaldehyde 15 min for endosomes
- in cold methanol for Golgi, 0.5% saponin 5 min
1% BSA in PBS 30 min
1o Ab stain (0.1% BSA) overnight at 4C
wash 30 min in PBS
2o Ab 1 h 37C
wash 30 min PBS
mount in DAPI
LAP2 (mouse)
LAP2 (mouse) kdorfman Tue, 10/02/2018 - 18:48Stains nuclear envelope
- fix in cold methanol
- 1:100
- BD transduction
- Mouse IgG
Lamin A/C
Lamin A/C kdorfman Fri, 11/04/2022 - 20:36mouse monocolonal (4C11)
7 ug/mL
Use at 1:100
(20 uL/class)
Storage:
- dilute 1:1 with glycerol,
- make 5 uL aliquots;
- add 245 uL PBS-Tw-Az-2% BSA
Lamin B1
Lamin B1 kdorfman Tue, 10/17/2023 - 16:30Rabbit anti-Lamin B1
Abcam AB16048
comes 100 µL at 1 mg/mL
Says to use at 0.1 µg/mL = 1:10,000 Drew's lab uses it at 1:1000
Make aliquots:
- 2 µL in a 2 mL tube
- label says
- 2 µL rabbit anti-lamin B1
- add 2 mL PBS-Tw-Azide
Aliquot and freeze - do not refreeze
Laminin (mouse)
Laminin (mouse) kdorfman Tue, 10/02/2018 - 19:01mouse anti-laminin gamma 1
stock concentration = 26 ug/mL
working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 80 uL ~12 mg/mL
Add 226 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Laminin (rabbit)
Laminin (rabbit) kdorfman Tue, 10/02/2018 - 19:08https://www.sigmaaldrich.com/catalog/product/sigma/l9393?lang=en®ion…
3 vials as of Spring 2018
dilute 1:25
266A freezer
Mucin (mouse)
Mucin (mouse) kdorfman Tue, 10/02/2018 - 19:03Monoclonal Anti-Mucin Gastric antibody produced in mouse
dilute 1:200 for use
dilute 1:1 in glycerol
Aliquot 4 uL; add 302 uL PBS BSA to make enough for 3 slides
Freezer 266A
Myosin (mouse)
Myosin (mouse) kdorfman Tue, 10/02/2018 - 18:46mouse anti-myosin light chain
stock solution: 37 ug/mL
working solution: 2 - 5 ug/mL
diluted 1:1 in glycerol
50 uL aliquots of ~18ug/mL
Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides
Freezer 266A
Myosin (rabbit)
Myosin (rabbit) kdorfman Tue, 10/02/2018 - 19:14Neurofilament (rabbit)
Neurofilament (rabbit) kdorfman Tue, 10/02/2018 - 19:16Nuclear pore complex
Nuclear pore complex kdorfman Wed, 10/18/2023 - 19:16Anti-Nuclear Pore Complex Proteins antibody (Mab414)
100 µL, 1 mg/mL
Dilute 1:1000 to use
2 µL aliquots. Label says:
- add 198 µL PBS-Tw-Az-BSA
- dilute again 1:10 for use
Mouse monoclonal [Mab414] to nuclear pore complex proteins
Suitable for ICC or IF
Reacts with mouse, human, Saccharomyces cerevesiae
Isotype: IgG
Store at 4C short term
Aliquot and freeze for long term storage
Fix in 100% methanol -20C OR 4% PFA in PBS pH 7.4 10 min RT
PKC (rabbit)
PKC (rabbit) kdorfman Tue, 10/02/2018 - 20:44PKC alpha (c-20)
rabbit polyclonal Abs 1gG
DO NOT FREEZE
In refrigerator 36sA
dilute 1:50 to use
Santa Cruz Biotechnology sc-208
From Wikipedia: Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes.[5]
Protein kinase C-alpha (PKC-α) is a specific member of the protein kinase family. These enzymes are characterized by their ability to add a phosphate group to other proteins, thus changing their function. PKC-α has been widely studied in the tissues of many organisms including drosophila, xenopus, cow, dog, chicken, human, monkey, mouse, pig, and rabbit. Many studies are currently being conducted investigating the structure, function, and regulation of this enzyme. The most recent investigations concerning this enzyme include its general regulation, hepatic function, and cardiac function.
Phospho H3 (rabbit)
Phospho H3 (rabbit) kdorfman Mon, 08/19/2024 - 17:55For showing mitotic cells
Histone H3 phosphorylation on serine-10 is specific to mitosis and phosphorylated histone H3 (PHH3) proliferation markers (as counts defined per area or as indices defined per cell numbers) are increasingly being used to evaluate proliferation in various tumors.
Dilute 1:2000
Synaptic vesicle (mouse)
Synaptic vesicle (mouse) kdorfman Tue, 10/02/2018 - 18:57Synaptic vesicle glycoprotein 2A
Stock concentration = 36 ug/mL
Working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 60 uL ~18 mg/mL
Add 246 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Troponin (mouse)
Troponin (mouse) kdorfman Tue, 10/02/2018 - 18:58DSHB TI-4-s
IgG (mouse)
Stock concentration = 43 ug/mL
Working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 50 uL ~22 mg/mL
Add 256 uL PBS-Tw-BSA to make enough for 3 slides
266A Freezer
Tubulin alpha (rat)
Tubulin alpha (rat) kdorfman Tue, 10/02/2018 - 16:23Accurate Chem YSRTmcADg YSRTMCA77G
Clone YL 1/2
Antibody as purchased (2019) was diluted 1:1 with glycerol
As purchased, should be diluted 1:200; glycerol-diluted AB should be diluted 1:100 with PBS-Tw-Az-BSA
Each tube has 4 µL antibody
Add:
400 µL PBS-Tw-Az-BSA
=404 µL, enough for 8 reactions (50 µL per coverslip, 60 min 37C)
Incubate coverslip on 50 uL antibody 1 hr 37C in humid chamber
Tubulin gamma (mouse)
Tubulin gamma (mouse) kdorfman Tue, 10/02/2018 - 17:17binds to centrosomes
From Pat
Monoclonal, Sigma clone GTU-88
Fix in paraformaldehyde or cold methanol
Dilute 1:100
From Drew Sigma T6557-100UL
- Working concentration is 1:5000
- Frozen stock is diluted 1:1 with glycerol
- 5 uL aliquots (in half-mL tubes) should be diluted:
- 1:50 in the tube: add 245 uL PBS-Tw-BSA to make 1:100 stock
- 1:50 for use (1 uL 1:100 antibody + 49 uL PBS-Tw-Az-BSA per coverslip)
Drew's Protocol:
- Rinse in PBS
- Fix in paraformaldehyde 15 minutes
- Rinse with PBS-Triton
- Rinse with PBS-Tween
- Rinse with PBS-BSA 1 hour
- Dilute Ab 1:5000 in BSA
- Incubate with Ab 2hr RT
- Rinse with PBS 3x
- Incubate with secondary Ab (in BSA)
- Rinse with PBS 3x
- Mount
Test Protocol:
- coverslips already fixed in paraglut, stored in PBS-Tw-Az 1
- 1 uL Ab (as supplied by Sigma) into 99 uL PBS-BSA (save!)
- 1 uL 1:100 Ab into 499 uL PBS-BSA (save for the rest of the slides if it works!)
- 50 uL onto coverslip - 2 hr RT incubation
- rinse with PBS-Tw-Az
- 50 uL Goat anti mouse 488
-
Results: some background fluorescence. Add a blocking step:
rinse 30 min in 2% BSA
rinse 2x in PBS-Tw-Az
Didn't make much difference. centrosomes are small and bright ↩︎
Vinculin (mouse)
Vinculin (mouse) kdorfman Tue, 10/02/2018 - 18:44Membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton, associated with cell-cell and cell-matrix junctions, where it is thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane.
Sigma V4505 monoclonal mouse
Fix in Formaldehyde
Dilute 1:100
ZO1 (Rabbit)
ZO1 (Rabbit) kdorfman Tue, 10/02/2018 - 16:24large tube has 10 µL antibody
small tube has 5 µL antibody
use at 1:100
362A freezer
fix in paraformaldehyde
Secondary antibodies
Secondary antibodies kdorfman Tue, 10/02/2018 - 19:19Goat anti-mouse IgM TRITC
Goat anti-mouse IgM TRITC kdorfman Tue, 10/02/2018 - 19:26Fisher OB102102 (Southern BioTech 1021-02)
Goat Anti-Mouse IgM (micron chain specific)-TRITC
1 mg/mL
1:100 - 1:400
1 µL aliquots. Add 100 - 400 µL 1% BSA
Use 50 µL per coverslip
Goat anti-mouse 488
Goat anti-mouse 488 kdorfman Tue, 10/02/2018 - 20:37Ex: 488 nm
Em: 499 nm
Stock(as purchased) = 2 mg/mL
Stock bottle has been diluted with glycerol: ~800 uL of 1 mg/mL
Working concentration = 1 ug/mL
Aliquots are 1 uL of 1 mg/mL
Add 611 uL to make enough for 6 slides, or up to 1 mL
Freezer 266A
Goat anti-mouse TRITC
Goat anti-mouse TRITC kdorfman Tue, 10/02/2018 - 19:23Anti-Mouse IgG (whole molecule) TRITC conjugate
Sigma T 5393
26 mg protein/mL
minimum working dilution = 1:64
1 µL aliquots. Add up to 100 µL 1% BSA
Alexa Fluor 568 (A11004) (red) 2mg/mL
working concentration ~10µg/mL
dilute 1:200
aliquots are 110 µL 20µg/mL
add
110 µL 2% BSA in PBS
to make 220 µL 10µg/mL
Use 50 µL per coverslip (there is enough for 4 per tube)
Goat anti-rabbit 546
Goat anti-rabbit 546 kdorfman Tue, 10/02/2018 - 20:39Stock concentration = 2 mg/mL
Working concentration = 4 ug/mL
Em = 575 nm
Ex = 560 nm
ALL OUT as of Spring 2018
Goat anti-rabbit FITC
Goat anti-rabbit FITC kdorfman Tue, 10/02/2018 - 20:42Goat anti-rabbit red
Goat anti-rabbit red kdorfman Tue, 10/02/2018 - 19:25Jackson Immuno 111-165-003
Cy3 Ex - 550 nm, em= 570 nm ("Red")
tubes : 2 µL. Add 398 µL PBS-Tw-Az to make 1:200
Use 50 µL per coverslip (there is enough for 4 per tube)
Goat anti-rat FITC
Goat anti-rat FITC kdorfman Tue, 10/02/2018 - 19:24Sigma F6258
Each tube has 3 µL antibody
Add
192 µL PBS-Tw-Az
195 µL 2% BSA
=390 µL, enough for 7 reactions (50 µL/coverslip 37C 30min)
Goat anti-rat TRITC
Goat anti-rat TRITC kdorfman Tue, 10/02/2018 - 19:31From Sigma AP136R EMB Millipore
ab peak = 550nm; emission peak = 570 nm
Bottle contains 2 mg. Add 1 mL H2O + 1 mL glycerol to make 1 mg/mL
aliquot 2 µL or 5 µL antibody, freeze
add 198 µL or 495 µL PBS-Tw-BSA
use 50 µL per coverslip, diluted to 1/100 (Try 37C 30 min)