CIM Antibody Core
F.A.Q.
Frequently Asked Questions

2. Transfer protein onto a Shleicher & Schuell Nitrocellulose membrane (0.4 u) 1 X Transfer Buffer with a BioRad Semi-Dry Transferring Cell 1 h at 12 V.

3. Block in 1X TBST containing 5% milk for 1 h at RT or ON at 4oC with agitation. .

4. Add anti-serum to a proper amount of 1 X TBST containing 5% milk (enough to cover the membrane) at 1:2000 or 1:1000 dilution. Shake the blot 1 to 2 h at RT.

5. Wash the blot with 1X TBST 3 times. Every time 5 minutes at RT.

6. Incubate the blot with PIERCE Immuno Pure Rabbit Anti-mouse IgG + IgM (H + L), HRP Conjugated, at a 1:5000 dilution for 1 h at RT.

7. Wash 3 times as step 5.

8. Incubate the blot with SuperSignal West Pico Chemiluminescent Substrate (Product # 34080) as described by the manufacturer.


Buffers:

10 X HEPES Running Buffer (1L):
    121g Tris
    238g Hepes
    Dissolve in 1 L H2O
    Add 10 g SDS

10 X TBST (10 L):
    121 g Tris, dissolved in 4 L H2O
    Adjust pH to 8.0
    Add 800 g NaCl
    Add 100 ml Tween-20
    Fill H2O to 10 L

10 X Transfer Buffer (1L):
    30.3 g Tris
    144 g glycine
    Dissolve in 1 L H2O. pH should be 8.3 (do not adjust)

To make 1 X, add Methanol to 10%