20150305 Set up Dot_Blot human_nerve_lysate + NFASC155_transfected_HEKs

Objectives

We want to know if we can see positive marks in positive patients sera with human nerve.

Material

• lysis buffer:
  • 0.87g NaCl
  • 200ul EDTA 0.5M
  • 20ml TRIS 0.5M
  • 0.5g deoxycholic acid
  • Up to 100ml with H2O
  • pH: 7.5
  • proteases inhibitor 10ul
• Human nerve
• NFASC155  transfected HEKs
• Patients sera
  • HVH1
  • LAC
  • Ctrl
  • Ctrl
  • JVMG
  • TPP
  • 1664/3
• Antibodies
  • NFASC155
  • GAC-680
  • GAH-800
• Blocking Odissey

Methods

1. Lysate human nerve with lysis buffer
2. Centrifuge 200ul of lysate
3. Put 5ul from the supernatant in each membrane
4. Put 5ul of transfected HEKs (NFASC155) (lysate) also in each membrane creating 2 dots in the membrane
5. Let the drops dry (aprox. 10min)
6. Incubate the membranes with blocking odissey 1:1 in PBS 1h in agitation
7. Incubate with patients sera or priamry antibody 2h in agitation:
   1. HVH1 1/100
   2. LAC 1/100
   3. Ctrl 1/100
   4. Ctrl 1/100
   5. JVMG 1/100
   6. TPP 1/100
   7. 1664/3 1/100
   8. chicken-anti-NFASC 1/2000
   9. chicken-anti-NFASC 1/2000
8. Wash 3 times with PBS 5minutes in agitation
9. Incubate secondary antibody 1h in agitation:

1-7. GAH-800 1/7500

8+9. GAC-680 1/7500

10. Wash 3 times with PBS 5 minutes in agitation

11. Scan the membranes with Odissey scanner

Results

We can only see positive marks for LAC with transfected HEKS but not with the lysate of human nerve.

Conclusions

We could try to put more lysate in the membrane trying to increase the quantity of the protein of interest.

Also we’ll try with FXM which is a patient with antibodies against PMP2, that is a more abundant protein.