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Protocols

ReliaBLOT® IP/Western Blot Reagents and Protocol

 

Cat. No. WB120 has been discontinued. See Reagents required for replacing contents included in kit.

ReliaBLOT® IP/Western Blot Reagents and Protocol provide an improved method for the detection of immunoprecipitated proteins assayed via Western blot.

IMPORTANT

Rabbit Antibodies For use with antibodies raised in rabbits.
Reduction For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels.
Heavy Chain When the ReliaBLOT® Reagents and Protocol are properly used, background from heavy chain should be reduced to less than 1% of that seen with standard procedures.
Sensitivity Sensitivity is correlated to the concentration of primary antibody used in the western blot. When performing western blots on immunoprecipitated samples, it is recommended to use the primary antibody 10-25 times more concentrated than that used in western blots on lysates. For example an antibody used at a dilution of 1:5000 in western blots on lysates might be used at a dilution of 1:500 in western blots on immunoprecipitated samples.
Reagents Both the Blocking Solution and the Goat anti-Rabbit Light Chain HRP Conjugate must be used.

 

Reagents required:

For in vitro laboratory use only. Not for any clinical, therapeutic, or diagnostic use in humans or animals. Not for animal or human consumption.

Suggested Protocols:

(Formulations for suggested buffers are included below.)

Preparation of Cell Lysate used for Immunoprecipitation:

Immunoprecipitation Reaction:

Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels.

Electrophoresis in SDS-Polyacrylamide Gel and Transfer to Nitrocellulose:

  1. Cut open the package that contains the gel cassette and drain away the buffer.
  2. Rinse the wells with distilled water.
  3. Rinse the wells with fresh running buffer.
  4. Place the gels on the buffer core so that the shorter plates face inward. If only using one gel, use a buffer dam to seal the other side.
  5. Fill the inner core with fresh running buffer. If there are no leaks, fill the outer core with running buffer. Loads samples.
  6. Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes).
  7. Soak nitrocellulose membrane and blotting paper in transfer buffer for at least 5 minutes prior to opening gel cassette.
  8. Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper.
  9. Place in transfer apparatus and fill with fresh transfer buffer.
  10. Run transfer apparatus for 60-75 minutes on 35V.

Western Blotting:

  1. Prepare Blocking Solution:  Add 19 g of non-fat milk and 20 ml of pig serum (Bethyl, Cat. no. S100-020) to 280 ml of TBST. This should be done approximately 30 minutes prior to use to ensure the powder dissolves completely. Store unused portion at 4° C. The resulting solution should be sufficient for thirteen Western blots (20 ml for step 2, and 15 ml each for steps 3 and 6 below).
  2. Remove the membrane from the transfer apparatus and place in 20 ml of Blocking Solution for one hour, with gentle shaking.
  3. Dilute the primary antibody in 15 ml of Blocking Solution. When using affinity purified antibodies, the recommended dilution for blotting immunoprecipitates is 1 µg/ml. (If using neat sera or an IgG fraction such as Protein-A purified antibody, higher concentrations are likely to be required. For best results, the optimal concentration of antibody should be empirically defined.)
  4. Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
  5. Wash the membrane three times, 10 minutes each time in TBST. 
  6. Dilute the Goat anti-Rabbit Light Chain HRP Conjugate (Bethyl, Cat. no. A120-113P) in 15 ml of Blocking Solution. It is recommended that the HRP Conjugate be used at a dilution of 1:1,000 to 1:10,000. For best results, the optimal concentration of HRP Conjugate should be empirically defined.
  7. Incubate the membrane in diluted Goat anti-Rabbit Light Chain HRP Conjugate for 60 minutes.
  8. Wash as directed in step 6.
  9. Develop blots with substrate solution and place in plastic membrane protector.
  10. Expose membrane to film or CCD camera.

Formulations for Buffers

PBS (Phosphate Buffered Saline, pH 7.2)

NaCl 8.0 g
KCl 0.2 g
NaH2PO4 0.23 g
Na2H PO4 0.12 g

Adjust the volume to 1 liter with distilled water.

Store at 4-25°C.

Packets for 1 liter of PBS are available from Sigma (Cat# P3813)

TBST (Tris Buffered Saline with Tween 20, pH8.0)

Tris 6.1 g
NaCl 8.68 g
Tween-20 500 µl

Adjust the volume to 1 liter with distilled water. Adjust pH to 8.0 with HCL.

Store at 4-25°C.

IP Lysis Buffer

NaCl 7.31 g
1 M Tris, pH 8 25 ml
0.5 M EDTA, pH 8 5 ml
10% NP-40 25 ml
Distilled Water 445 ml

Store at 4°C.

Protein -A Agarose Beads
Protein A Sepharose Beads are available from Amersham (Cat# 17-0780-01). To prepare the beads, swell 400 mg of beads in 10 ml of distilled water for 30 minutes. Centrifuge at 200 x g for 60 seconds. Remove the supernatant and replace with 10 ml cell lysis buffer. Centrifuge as above, remove the buffer and replace with 10 ml cell lysis buffer. Repeat this step three times, for a total of 4 washes with cell lysis buffer. After the last wash add 8 ml of cell lysis buffer. The resulting slurry an approximately 20% slurry of beads. Store at 4°C for one month. Discard after one month.

4X Sample Buffer

Glycerol 4.0 g
Tris Base 0.68 g
Tris HCL 0.67 g
LDS 0.80 g
EDTA 6 mg
Brilliant Blue G250 2.5 mg
Phenol red 2.5 mg

Adjust volume to 10 ml with ultra pure water.

Store at 4°C.

DTT Reducing Agent

DTT (Sigma Cat# D-0632) 1.54 g
Ultra pure water 10 ml

Aliquot and store at -20°C. At time of use, thaw an aliquot. Discard excess. Do not refreeze.

1X Sample Buffer

4X sample buffer 150 µl
1M DTT 60 µl
Distilled water 390 µl

Make fresh for each use.

10X Transfer Buffer

Tris (free base) 15.2 g
Glycine 72.1 g
SDS 5.0 g

Ultra pure water to 500 ml

Store at 4°C.

1X Transfer Buffer

10X Transfer Buffer 50 ml
Methanol 100 ml
Distilled water 350 ml

Make fresh for each use.

20X Running Buffer

Tricine (free base) 71.7 g
Tris (free base) 72.6 g
SDS 10.0 g
Sodium Bisulfite 2.5 g

Adjust to 500 ml with ultra pure water.

Store at 4°C. For 1X Running Buffer, add 10 ml of 20X Running Buffer to 190 ml of distilled water.