Hello,

we're trying to detect Keratin 14 and 15 in Keratinocytes as well as FN-1 and CD90/THY1 in Fibroblasts from human skin. As reference gene GAPDH and Vinculin are used.

In our first experiments we used primary fibroblasts. We were only able to detect GAPDH and did not get any signal from CD90/THY1 or FN-1. Furthermore, we used HaCats for antibody titration of Keratin 14 and 15. You can see the presumed bands attached.

Our set-up:

Protein extraction

Protein extraction buffer: 50 mM Tris-HCl, pH 7,7; 1 % Triton X-100; 1 mM Dithiothreitol (DTT)

1. Addition of 75 µl protein extraction buffer each (samples were pellets in 1,5 ml reaction tubes from cells that were trypsinized and centrifuged before; cell count: approx. 2-3 x 10^6 cells)

2. 40 s ultrasound

3. 2 min in liquid nitrogen

4. Thaw in thermomixer at 37 °C Duration: 2 min

5. Repeat steps 3. and 4.

6. Centrifuge the lysates at 4 °C; max. rpm for 4 min

7. Transfer supernatant into eppi

8. Store supernatants at -20 °C

SDS-PAGE

precast gels from Bio-Rad

1-fach TGE (25 mM Tris; 250 mM Glycin, pH 8,3; 0,1 % SDS)

Loading with 5, 10, 15 and 30 µg protein sample

1. Mixing of Lämmli buffer and samples 1:1

2. Boil for 5 min at 95 °C

3. Loading gel

4. 100 V until separating gel separates

5. 200 V until the running front is completely run through

Western Blot (Tank blot)

Transfer buffer: Tris Base: 58,2 g; Glycine: 29,3 g; VE-Wasser ad 800 ml

1. Transfer unit, which is aligned to the negative pole (black)

2. Fibre mat (previously immerse in transfer buffer)

3. Filter paper (previously immerse in transfer buffer)

4. Gel from the SDS-PAGE → place on top as free of bubbles as possible

5. Nitrocellulose membrane → place on top as free of bubbles as possible

6. Filter paper (dip in transfer buffer beforehand)

7. Remove air bubbles; from the centre outwards each time

8. Fibre mat (previously immerse in transfer buffer)

9. Transfer unit, which is directed to the positive pole (white)

10. Transfer at 100 V for 1 h

Staining of membrane

1. Staining with Colloidal Gold Total Protein Stain for 1 h, Cutting the membrane

2. Rinse the membrane with 100 ml deionised water for 1 min, Repeat 2x with fresh water

3. Rinse with TBS

Immunoblot

1. Incubate the membranes in 5 % MP(milk powder)-TBST on a shaker for 45 min (blocking)

2. Incubation with primary AK in 1% MP-TBST for 60 min at RT on the shaker

3. Rinse 3 x 5 min with TBST

4. Incubation with secondary AK for 60 min at RT on the shaker

5. Rinse 3 x 5 min with TBST

Solution A → 100 ml

0.1 M Tris-Cl; pH 8.6 100 ml

Luminol (Sigma A4685): 25 mg

Solution B → 10 ml

para-hydroxycoumaric acid (Sigma C9008) 11 mg

DMSO 10 ml

ECL-Reagent: 4 ml solution A and 400 µl solution B + 1,2 µl H2O2

6. Place the membrane on the open autoclave bag

7. Lift membrane with tweezers → let TBST drain off

8. Pour ECL reagent over membrane and incubate for 2 min IN THE DARK.

9. Lift membrane and let ECL reagent drip off

10. Transfer membrane to new foil and detect chemiluminescence in the Lumi-Imager

11. Exposure Time 3 min

You can see the result in the pictures attached. You can only see the bands of GAPDH. The other blots are empty.

Does anybody have a suggestion or idea why we can only detect GAPDH in Fibroblasts (primary) or Keratinocytes (HaCat) but no other targets?

Hint: The expression of the targets has already been shown by qPCR.