Antibodies

Antibodies kdorfman Tue, 10/02/2018 - 16:21

For 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 - 16:21

5-mC (rabbit)

5-mC (rabbit) kdorfman Fri, 11/05/2021 - 13:44

5-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 - 18:50

DSHB JLA20

stock 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 - 19:04

Sigma A2668

Dilute 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 - 18:51

DSHB B4-78

stock 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 - 17:46

Binds 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 - 18:59

DSHB II-II6B3

stock 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 - 19:00

DSHB M3F7

mouse 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 (rat)

Connexin (rat) kdorfman Tue, 10/02/2018 - 19:17

DSHB R5.21C

stock 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 - 13:39

D63A6 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 - 17:11

1 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 - 16:23

Novus Biologicals, recombinant human Hec1, monocolonal
fix in methanol
dilute 1:200

Histone H3 phospho S10

Histone H3 phospho S10 kdorfman Fri, 11/04/2022 - 20:46

FINISH WORKING ON THIS!

Rabbit polyclonal

Abcam AB5176

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 - 19:18

DSHB Ralph 3.1

rat 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 - 16:22

GeneTex GTX13523

IgG

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

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

reference

LAP2 (mouse)

LAP2 (mouse) kdorfman Tue, 10/02/2018 - 18:48

Stains nuclear envelope

Lamin A/C

Lamin A/C kdorfman Fri, 11/04/2022 - 20:36

Cell Signalling 4777S

mouse 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

Laminin (mouse)

Laminin (mouse) kdorfman Tue, 10/02/2018 - 19:01

DSHB 2E8

mouse 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

Mucin (mouse)

Mucin (mouse) kdorfman Tue, 10/02/2018 - 19:03

Monoclonal Anti-Mucin Gastric antibody produced in mouse

Sigma M5293

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 - 18:46

DSHB T14

mouse 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 - 19:14

Sigma M7523

Formalin fixation

2 vials as of spring 2018

Dilute 1:20

freezer 266A

Neurofilament (rabbit)

Neurofilament (rabbit) kdorfman Tue, 10/02/2018 - 19:16

Anti-Neurofilament 200 antibody produced in rabbit

Sigma N4142

Dilute 1:80

Freezer 266A

PKC (rabbit)

PKC (rabbit) kdorfman Tue, 10/02/2018 - 20:44

PKC 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.

Synaptic vesicle (mouse)

Synaptic vesicle (mouse) kdorfman Tue, 10/02/2018 - 18:57

DSHB SV2

Synaptic 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 - 18:58

DSHB TI-4-s

Anti-troponin I

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 - 16:23

Accurate 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)

Tubulin gamma (mouse)

Tubulin gamma (mouse) kdorfman Tue, 10/02/2018 - 17:17

binds 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

  1. 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 - 18:44

Membrane-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 - 16:24

large 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 - 19:19

Goat anti-mouse IgM TRITC

Goat anti-mouse IgM TRITC kdorfman Tue, 10/02/2018 - 19:26

Fisher OB102102 (Southern BioTech 1021-02)

Goat Anti-Mouse IgM (micron chain specific)-FITC

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 - 20:37

Invitrogen A-11001

Ex: 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 - 19:23

Anti-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 FITC

Goat anti-rabbit FITC kdorfman Tue, 10/02/2018 - 20:42

Goat anti-rabbit IgG FITC

Sigma F0382

Dilute 1:80

Goat anti-rabbit red

Goat anti-rabbit red kdorfman Tue, 10/02/2018 - 19:25

Jackson 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 - 19:24

Sigma 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 - 19:31

From 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)