Using Bovine Serum Albumin (BSA) as a Blocking Agent

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Using Bovine Serum Albumin as a Blocking Agent

Western Blot with BSA as a Blocking Agent

Solutions:

10x Phosphate Buffered Saline—PBS (for removing methanol from blots)
NaCl 80g (1.37 M for 10x)
KCl 2g (27mM for 10x)
Na₂HPO₄(80mM for 10x)
KH₂PO₄(20mM for 10x)
water to 1L
pH to 7.4 with HCl

PBS Tween—PBST (for washing blots)
PBS 2L
Tween-20 (Brown bottle) +1mL by pipetting it in with pipettor or plastic bulb
stir half an hour

Protocol :

Blotting
1. Wet a dried, transferred blot with methanol if the blot is made with PVDF, or soak in PBS if blot is made from nitrocellulose (note that methanol is toxic, so wear gloves and don’t inhale it) FOR NITROCELLULOSE BLOTS DON’T WET WITH METHANOL, OR ELSE IT’LL DISTINTEGRATE IN YOUR HANDS
2. Rinse ~4x with PBS to get rid of the methanol
3. Place the blot face up for blotting
4. Block with 1-5% BSA for 10 min. to overnight (block at 4 degrees if desired) - block with 5% BSA and overnight if there is high background, and less if there is less background
5. Primary antibody incubation: 10mL total--Primary antibody diluted to the appropriate concentration in PBS (+0.2% BSA) 1hr
6. Washes: 3x 10min. each PBST
7. Secondary antibody incubation: 1:5000 dilution of the horseradish peroxidase secondary (choose the right secondary—either goat anti-rabbit horseradish peroxidase (HRP), or goat-anti mouse HRP depending on the type of primary antibody used), 30 minutes
8. More washes: ~5x PBST 5-10 minutes each
(Note that horseradish peroxidase is an enzyme and thus temperature sensitive, so it is best to leave it at 4 degrees if you can not immediately continue to develop it; however, try not to leave it at 4 degrees for too long [1-2 hours is probably OK])

Viewing the blot
1. mix equal amounts of solution A & B of ECL (~1.5mL each of A & B)
2. rock manually for 1 min.
3. wrap Saran Wrap around blots
4. take off gloves when handling film
5. turn off all lights (can leave a dim red light on) when removing film from the box, and place remaining film back into box before dealing with the newly-removed piece of film
6. place film on top of blot in the film cassette, making a note of the orientation of the film
7. expose for 15sec -2 minutes, and if required, use another film and expose for anther 20 min.
8. develop the blot

Immunofluorescence with BSA as a Blocking Agent

Solutions:

5x PB Buffer ( = 250 mM phosphate buffer, pH 7.2 after dilution)
To make 500mL,
Mix 140mL 0.25M NaH₂P0₄
+ 360mL 0.25M NaH₂P0₄
OR
1x PB, 500mL
35 mL 0.2M NaH₂P0₄
90 mL 0.2M NaH₂P0₄
375mL water
1x PB is required for the procedures below

20% Triton
· Weigh 2g Triton on a balance by pipeting into a tube,
· Make up to 10mL with distilled water
· Rock overnight

4% paraformaldehyde (STIR IN FUMEHOOD)
To make 50 mL,
· use ~40 mL water to dissolve 2g paraformaidehyde
· add 200uL 0.2N NAOH and heat, while stirring in the fumehood
· once dissolved, + 10mL of 5x PB buffer
· freeze in ~10-15mL aliquots in the –20 freezer

Normal Goat Serum—NGS
· store in aliquots in the freezer

Notes:
· if after fixing the cells, leave them wet in the fridge, covered with the PB from the wash
· be careful when pipeting and removing solutions; otherwise, cells will come off

Protocol

1. Wash 1 x with PB
2. Fix with 4% paraformaldehyde (10 min., 15-30niin. if cells easily come off)
3. Wash 3x with PB (5 min. each)
4. Preincubation: for every 1 mL PB, + 1-10% BSA, 10uL 20% Triton (15 min.)
5. Primary antibody: for every 1mL total volume, add 20uL BSA, 5uL triton (2hrs); dilution of primary varies with each antibody and must be tested empirically or according to manufacturer's directions
6. Wash 3x with PB (10 min. each)
7. Secondary antibody: choose the correct one (either goat anti-mouse, or goat anti-rabbit) CY3 (1/800 dilution): for every 1mL PB, 1.25uL secondary, 5uL triton, 25uL BSA (30 minutes-don't go too much longer, or else high background)
8. Wash 3x with PB (15 min. each)
9. Take out the mowial to thaw; take out cover slip, drain it a bit with Kleenex, +20uL mowial on the cells and cover the cover slip with it
10. Invert the cover slip on the slide
11. Place nail polish on the four corners of the cover slip. When the nail polish dries, put nail polish all around the slides of the cover slip.
12. You may view it or put it in the dark at four degrees. The slides may be stored in the dark at four degrees.

ELISA with BSA as a Blocking Agent

Materials

Nunc microtiter plates (medium binding) polysorp F96 475094 (RIA plates)
Maxisorp F96 442404

ABTS (Available at Sigma, for example)

Buffer A
Disodium hydrogen orthophosphate 1M (20mL water)
Citric Acid 1M (16mL water)
Combine and dilute to 164 mL, pH to 4.0
Store in the dark at room temperature

Notes

Do samples in triplicate
+60uL of each solution to the well each time; washes/rinses are applied to fill the well and removed by inverting the plate and flinging out excess solution
Note that all proteins (including BSA) use 1x PBS as the diluent
Variations include applying certain proteins using a serial or 10x dilution
Can use multichannel pipet to speed up delivery of solutions to microtiter plates

Procedure

- Plate Protein 1 overnight at 37 degrees in incubator (cover it with a 1mL pipet tip box) - dilute protein to 1 to 100 ug/mL
- Rinse with PBS
- Block with 4% BSA in PBS, 1 hour
- Rinse with PBS
- Add Protein 2 + 0.1% BSA
- Incubate 1-4 hours at room temperature or at 37 degrees Celsius
- Wash 3x with PBS-Tween
- Incubate 1 hour at room temperature with Protein 3 (if there is one) + 0.1% BSA
- Wash 3x with PBS-Tween
- Incubate 1 hour with room temperature with Protein 4 or antibody, which is conjugated to - peroxidase (dilute the peroxidase-conjugated protein or antibody 1/1000 with PBS-Tween) + 0.1% BSA
- Wash 5-6x with PBS-Tween
- developing solution (5mg ABTS in 10mL of Buffer A + 10uL H₂O₂) – make just before use and keep in the dark prior to use
- Wait 15 minutes (put foil around it to prevent light from reaching it)
- Read the microtiter plate with a plate reader at 405nm

For controls, omit each protein or BSA.
Another control could be a nonspecific primary antibody replacing the regular one, and BSA replacing Protein 1.