0: Pre-sem prep
0: Pre-sem prep kdorfman Wed, 12/07/2011 - 15:49Pre-semester prep
Check out 20 minute secondary antibodies from Invitrogen
Get 2 boxes each: 20 mm and 14 mm coverslip dishes from Invitro-Scientific, much cheaper than MatTek.
http://www.invitrosci.com/product_detail.php?product_id=21
http://www.invitrosci.com/product_detail.php?product_id=25
Nucleofector kit: Ingenio Electroporation kit, MIR50112
Fisher: MIR50112 $230.17. More expensive than mirusbio, but mirus charges $35 for shipping.
Solutions
Solutions kdorfman Tue, 12/27/2011 - 19:21Solutions to have at the beginning of the semester
Amount | conc | solution | |
---|---|---|---|
100 | mL | 10% | Tween in water |
50 | mL | 10% | Tween in PBS |
100 | mL | 10% | Triton-X in water |
100 | mL | 10% | Triton-X in PBS |
5 | L | 10X | PBS |
1 | L | 1x | PBS in 10 mL sterile aliquots |
4 | L | 1x | PBS-Tw-Az |
25 | mL | 10% | Sodium azide |
250 | mL | 10X | HBS |
100 | mL | 10 X | Ca-free HBS |
500 | mL | 1x | Non-CO2 medium with serum |
1000 | mL | 1x | Non-CO2 serum free medium |
2 | L | 1x | F10-Hams |
250 | mL | 1x | serum free F-10 Hams |
1 | L | 1x | DMEM |
20 | mL | DAPI Fluoromount Fisher OB010020 2 bottles to share. |
Aliquots
Aliquots kdorfman Tue, 12/27/2011 - 19:27Students use lots of these:
- 10 mL PBS
- 10 mL serum-free-non-CO2 medium
- 10 mL HBSS
- 10 mL HBS
Slides
Slides kdorfman Tue, 12/27/2011 - 20:56slides 2012
slides 2012 kdorfman Fri, 08/23/2013 - 14:07CHECK THIS
LLCPk-1
coverslips, fixed in 3.5% formaldehyde and stored in PBS-tween-azide in coplin jars in the refrigerator:
30 unmounted,
10 mounted, stained only with DAPI
20 stained for tubulin, and mounted with DAPI
20 stained for actin and tubulin, and mounted with DAPI
10 serum starved, stained for tubulin, mounted with DAPI
- for Lab 4.1
- Grow first in regular medium (otherwise they won't stick)
- Replace medium with serum free medium.
- Fix after 24 hours
10 Arrested with 100nM nocodazole (8 - 16 hours)
- for Lab 4.1
- Treat with nocodazole late in the day
- Fix the next day in para-glut or formaldehyde
- Stain for tubulin, mount with DAPI
Nocodazole stock is 33 mM in DMSO
Make 100 µL 1 mM: 3 µL (33mM) + 97 µL medium
Make 1 mL 6µM: 6 µL (1mM) + 994 µL medium
Add 1 drop (~50 µL) 6µM to each dish of ~3 mL
OR
Make 30 mL 100 nM: 3 µL (1mM) in 30 mL medium
Change the medium in each dish.
3t3
12 for Lab 3.3, unmounted
slides 2013
slides 2013 kdorfman Fri, 08/23/2013 - 14:08Lab | date | # | DAPI + | fix |
---|---|---|---|---|
1.1 & 1.3 | 9/4 & 11 | 10 | paraglut | |
1.2 | 9/9 | 10 | alpha tub | paraglut |
2.1 & 2.2 | 9/16 | 10 | alpha tub, actin | paraglut |
3.1 | 9/23 | 10 | alpha tub, actin | paraglut |
3.2 | 9/25 | 2 | Golgi | formaldehyde (no glut) |
2 | gamma tubulin | paraglut or methanol | ||
2 | actin | paraglut | ||
2 | alpha actinin | formaldehyde (no glut) | ||
2 | ZO1 | methanol or paraformaldehyde | ||
2 | LAP2 | methanol | ||
2 | vinculin | formaldehyde |
slides 2015
slides 2015 kdorfman Tue, 07/14/2015 - 14:58LL's for student slides
Fix 90 in formaldehyde; 10 in methanol
Plate 102 in 17 6-well plates.
Lab | # coverslips | fix | stain | secondary |
---|---|---|---|---|
1.1 | 10 | form or paraglut | ||
1.2 | 10 | " | ||
1.3 | 10 | " | ||
2.1 | 10 | " | anti-tubulin, phalloidin | goat anti-rat |
2.2 | 10 | " | anti-tubulin | goat anti-rat |
3.1 | 10 | " | anti-tubulin, phalloidin FRESH | goat anti-rat |
3.2 | 3 | form | alpha actinin | anti-mouse IgM |
3.2 | 3 | form | anti Golgi | anti-mouse IgG |
3.2 | 3 | form | vinculin | |
3.2 | 3 | MeOH | anti gamma-tubulin | |
3.2 | 3 | " | anti-Hec1 | |
3.2 | 3 | " | LAP2 | |
3.3 | 20 | form or paraglut | un-mounted for students to stain |
organelle slides
organelle slides kdorfman Tue, 07/14/2015 - 14:59Alpha actinin
Alpha actinin kdorfman Tue, 07/14/2015 - 15:02Stains adhesions and stress fibers
- Monoclonal, BM 75.2
- Fix in Formaldehyde, no glut or meOH
- 1:200
- IgM, use mouse FITC secondary that recognizes IgMs
Gamma tubulin
Gamma tubulin kdorfman Tue, 07/14/2015 - 15:03Stains Centrosomes
- Monoclonal, GTU-88, Sigma T6557
- Fixation: methanol; para/glut
- Dilution 1:100
From Sigma:
General description
γ-Tubulin (48kDa) is a ubiquitous and highly conserved protein within the microtubule organizing centers (MTOCs) in eukaryotic cells. γ-Tubulin is mapped to human chromosome 17q21.2 and codes for a member of the tubulin family. Human TUBG1 transcript is widely expressed in preimplantation embryos and brain. Monoclonal Anti-γ-Tubulin (mouse IgG1 isotype) is derived from the GTU-88 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse.
Specificity
Monoclonal Anti-γ-Tubulin recognizes an epitope located in the N-terminal amino acids of γ-tubulin (48 kDa). Cross reactivity has been observed with human, bovine, dog, hamster, rat, mouse, chicken, and Xenopus γ-tubulin. The antibody recognizes an epitope located within the N-terminal region of γ-tubulin.
Immunogen
synthetic γ-tubulin peptide, conjugated to KLH
Application
Monoclonal Anti-γ-Tubulin antibody has been used in western blotting,[1] indirect immunofluorescence (IF) and immunofluorescence staining. Monoclonal Anti-γ-Tubulin is also suitable for use in immunochemical applications such as immunoblotting, immunocytochemical staining of cultured cells and in ELISA. Monoclonal Anti-gamma-Tubulin is suitable for use in immunochemical applications such as immunoblotting, immunocytochemical staining of cultured cells, and in ELISA.
Biochem/physiol Actions
γ-Tubulin nucleates microtubule assembly throughout the mammalian cell cycle in vivo. γ-Tubulin binds microtubule minus ends and is responsible for mediating the link between microtubules and the centrosome. It functions as the microtubule nucleator at the MTOC. It binds to the β-tubulin half of the tubulin molecule, thus establishing the polarity of a microtubule, leaving the α-tubulin half exposed at the plus end. γ-Tubulin abundance is less than 1% of the level of either α- or β-tubulin. Overexpression of γ-tubulin is observed in lung cancer. The expression levels of γ-tubulin can be considered as an important prognostic indicator for patients with astrocytomas.
Physical form
The product is provided as ascites fluid with 15 mM sodium azide as a preservative.
Storage and Stability
For continuous use, store at 2-8 °C for up to one month. For extended storage, freeze in working aliquots. Repeated freezing and thawing, or storage in "frostfree" freezers, is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use.
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Golgi 58K
Golgi 58K kdorfman Tue, 07/14/2015 - 15:04- Sigma G2404, mouse monoclonal
- Formaldehyde (no glut)
- 1:100
Hec1
Hec1 kdorfman Tue, 07/14/2015 - 15:05Stains kinetochores
- Novus biological, recombinant human HEc1, monoclonal
- Fix in methanol
- 1:200
LAP2
LAP2 kdorfman Tue, 07/14/2015 - 15:07Stains nuclear envelope
- Methanol
- 1:100
- BD transduction
- Mouse IgG
Vinculin
Vinculin kdorfman Tue, 07/14/2015 - 15:08Focal adhesion plaques
- Monoclonal, sigma V4505
- Formaldehyde
- 1:100