20150304 Set up Dot_Blot NFASC155_transfected_HEKs

Objectives

We want to know if by dot blot we can see positive marks from positive patients sera.

Materials

• Patients sera:
  • HVH1
  • LAC
  • Ctrl14
  • Ctrl9
  • PVC
  • TPP
  • JML
• Antibodies:
  • Chicken-anti-neurofascin
  • GAH-800
  • GAC-680
• Blocking odissey
• PBS

Methods

1. Put 5ul of non-transfected HEKs (lysate) in each membrane
2. Put 5ul of transfected HEKs (NFASC155) (lysate) also in each membrane creating 2 dots in the membrane
3. Let the drops dry (aprox. 10min)
4. Incubate the membranes with blocking odissey 1:1 in PBS 1h in agitation
5. 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. PVC 1/100
   6. TPP 1/100
   7. JML 1/100
   8. chicken-anti-NFASC 1/2000
   9. chicken-anti-NFASC 1/2000
6.  Wash 3 times with PBS 5minutes in agitation
7. Incubate secondary antibody 1h in agitation:

1-7. GAH-800 1/7500

8+9. GAC-680 1/7500

8. Wash 3 times with PBS 5 minutes in agitation

9. Scan the membranes with Odissey scanner

Results

We see positive marks for those membranes incubated with comercial antibody and those incubated with LAC or JML.

Conclusions

This technique is adequate to see positive marks in positive patients with transfected cells.

Now, we need to know if we can see positive marks with a lysate of human nerve, in order to select candidate patients for IP.