Salivary Gland Extraction For Western Blot
updated 10/16/05 - Benjamin Costantino
(For the PDF version, click here.)
Required Supplies:
1x complete DPBS
Lysis Buffer
Protease Inhibitor Cocktail (Sigma P-8340)
2x lamellae
dissecting tools
500µl microfuge tubes
microscope slide
Solutions:
2x Salts
3.04g NaCl (104 mM)
2.98g KCl (80 mM)
2.38g HEPES (20 mM)
0.30g MgSO4 x 7H20 (2.4mM)
0.24g MgCl2 x 6H20 (2.4mM)
0.28g Na2HPO4 (4mM)
0.054g KH2PO4 (0.8 mM)
Water to 500 mL
Aliquot in 25 mL lots and freeze at -20°C
1x Complete DPBS (50 mL)
25 mL 2x salts
1.54g sucrose (90 mM)
0.09 g glucose (10 mM)
CaCl2 to 1mM
Water to 50 mL
pH to 7.2
Lysis Buffer
1% NP-40
0.5% Deoxycholic acid
0.1% Triton-X-100
100 mM NaCl
0.1 mM CaCl2
2 mM MgCl2
water to preferred amount
pH 7.6
(can be stored up to 1 year)
2 x Lamellae
add 50µl BME (betamarcaptoethanol) to 2x lamellae tube
stored in aliquots in -20°C
Procedure:
1. Aseptically transfer ~20mL of complete 1x DPBS into a sterile eye dropper or tube.
2. Fill 3 wells of glass dissecting dish with DPBS to provide a wash for larvae.
3. Remove larvae and place them in the wash to clean food and debris off of them.
4. On a clean microscope slide add 1 drop of DPBS.
5. Dissect animals in your preferred technique.
6. Transfer the glands to a 500µl microfuge tube containing 50µl of DPBS.
7. Continue collecting until you have 10-20 glands. **
8. Spin tube full speed for 1 minute to pellet glands.
9. Remove DPBS without disturbing gland pellet.
10. Add 50µl of lysis buffer containing protease inhibitor cocktail (PIC).
Prepare lysis buffer/protease inhibitor cocktail as follows:
- separate Protease Inhibitor Cocktail (PIC) into 10µl aliquots.
- store aliquots of PIC in -20°C.
- transfer 990µl of lysis buffer into one tube of 10µl PIC aliquot.
11. Disturb the pellet by tapping the tube.
12. Spin tube at high speed for 1 minute.
13. Aspirate old buffer with drawn-out Pasteur pipette, being careful not to disturb the pellet.
14. Add 10-20µl of lysis buffer (10pairs/10µl lysis buffer + 10µl of lamellae).
15. Homogenize glands with pestle made from heated tip of Pasteur pipette.
16. Add 10-20µl of 2x lamellae to tube with homogenized salivary glands.
17. Seal tube using heated Pasteur pipette and heat at 95°C for 5 min.
18. Store at -20 or -80°C for no more than a week before running gel.
Notes
* Washing once for a minute doesn't seem to affect anything.
** Generally 10 pairs in 20µl complete buffer [10µl lysis buffer + 10µl of 2x lamellae])
