Using Bovine Serum Albumin (BSA) as a Blocking Agent

10x Phosphate Buffered SalinePBS (to remove methanol from spotting)
80g NaCl (1.37 M to 10x)
2g KCl (27mm to 10x)
Na2HPO4 (80mm to 10x)
KH2PO4 (20mm to 10x)
water to 1L
pH 7.4 with HCl

PBS TweenPBST (for washing blots)
2L PBS
Tween-20 (Brown bottle) + 1mL by Pipetting in the pipettor or plastic balls
stir half an hour

protocol:

blotting

  1. Wet Dry, transferred blot with methanol if the blot made with PVDF, or soak in PBS if made of nitrocellulose blot (note that methanol is toxic, so wear gloves and Dont inhale it) nitrocellulose blots DON T FOR WET WITH METHANOL, OR itll DISTINTEGRATE ELSE IN YOUR HANDS
  2. Rinse ~ 4x with PBS to remove methanol
  3. Place face blot for blotting
  4. Block with 1-5% BSA for 10 minutes. for overnight (block at 4 degrees if desired) – blocks with 5% BSA and overnight if there is a high background, and less if there is less background
  5. The primary antibody incubation: 10 mL total – the primary antibody diluted to appropriate concentration in PBS (+ 0.2% BSA) 1 hour
  6. Wash: 3x 10 minutes. each PBST
  7. Secondary antibody incubation: 1: 5000 dilution of horseradish peroxidase secondary (right pick secondaryeither goat anti-rabbit horseradish peroxidase (HRP), or goat anti-mouse HRP antibody depends on the type of primer used), 30 minutes
  8. Further washing: ~ 5x PBST 5-10 minutes each
    (Note that the horseradish peroxidase enzyme and thus are sensitive to temperature, so it is best to leave it at 4 degrees if you can not immediately continue to develop it, but try not to leave it at 4 degrees too long [1-2 hours probably OK])

Seeing blot

  1. Mix equal amounts of solution A and B of ECL (~ each A & B 1.5ml)
  2. stone manually for 1 minute.
  3. around spotting wrap Saran Wrap
  4. take off the gloves when handling Film
  5. Turn off all the lights (can leave a dim red light on) when removing the film from the box, and place the remaining film back into the box before dealing with the newly-removed pieces of the film
  6. Place the film on top of the blot in the film cassette, make a note of the orientation of the film
  7. expose to 15sec -2 minutes, and if necessary, use another film and expose for anther 20 minutes.
  8. develop blot

Immunofluorescence with BSA as Blocking Agent

solution:

5x Buffer PB (= 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 of 0.2 M NaH2P04
    90 mL of 0.2 M NaH2P04
    water 375ml
    1x PB required for the procedure below

20% Triton
Weigh 2g Triton on balance by pipeting into the tube,
Make up to 10 ml with distilled water
stone overnight

4% paraformaldehyde (STIR IN chimney)
To make 50 mL,
Using ~ 40 mL of water to dissolve 2g paraformaidehyde
add 200ul 0.2N NaOH and heat, while stirring in the chimney
once dissolved, + 10 mL 5x buffer PB
~ 10-15mL frozen in aliquots at 20 freezer

Normal Goat SerumNGS
store in aliquots in freezer

Note:
if after repair cells, leaving them wet in the fridge, covered with a PB of washing
be careful when pipeting and removing the solution; if not, the cells will come from

Protocol

  1. Wash 1 x with PB
  2. Fix with 4% paraformaldehyde (10 min., 15-30niin. If the cells easily come from)
  3. Wash 3x with PB (5 min. Each)
  4. preincubation: for each 1 mL PB, + 1-10% BSA, 10ul of 20% Triton (15 min.)
  5. Primary antibody: for each total volume 1mL, add 20uL BSA, triton 5uL (2hrs); primary dilution varies with each antibody and must be tested empirically or in accordance with the direction of the manufacturer
  6. Wash 3x with PB (10 min. Each)
  7. Secondary antibody: selecting the correct (either goat anti-mouse or goat anti-rabbit) Cy3 (1/800 dilution): for each 1 mL PB, secondary 1.25uL, 5uL triton, 25uL BSA (30 min-don ‘ t go too long, or else high background)
  8. Wash 3x with PB (15 min. Each)
  9. Remove mowial for disbursement; take insurance slip, drain bit with Kleenex, + 20uL mowial on cells and covered with a glass cover.

ELISA with BSA as Blocking Agent

ingredient

Nunc microtiter plates (binding media) 475 094 F96 polysorp (RIA plates)
Maxisorp F96 442 404

ABTS (Available in Sigma, for example)

buffer A
1M disodium hydrogen orthophosphate (20ml water)
1M citric acid (16mL water)
Combining and dilute 164 ml, pH to 4.0
Store in the dark at room temperature

record

Are samples in triplicate

  • 60uL each time the solution to each well; wash / rinse applied to fill the well and removed by inverting the plate and throw out the excess solution

Note that all proteins (including BSA) using 1x PBS as diluent

Variations include applying a particular protein or 10x using serial dilutions

Can use a multichannel pipette to accelerate the delivery of solutions for microtiter plates

Procedure

  • Plate Protein 1 overnight at 37 degrees in the incubator (cover with pipette tip box 1mL) – dilute protein to 1 up 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 any) + 0.1% BSA
  • Wash 3x with PBS-Tween
  • Incubate 1 hour at room temperature with 4 protein or antibody, the conjugate – a peroxidase (dilute peroxidase-conjugated protein or antibody 1/1000 with PBS-Tween) + 0.1% BSA
  • Wash 5-6x with PBS-Tween
  • developing a solution (5 mg in 10 mL ABTS Buffer A + 10ul H2O2) made just prior to use and kept in the dark before use
  • Wait 15 minutes (put foil around it to prevent light from reaching it)
  • Read microtiter plate with a plate reader at 405nm

To control, remove any proteins or BSA.
Other controls can be a specific primary antibody to replace the usual one, and replacing BSA Protein 1.

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