Help! Western Blot - (Mar/12/2003 )

Result of Western Blot
No any band appears for most times
Some times very weak band could be detected

Questions:
Is the loaded sample not enough for detection by Western?
Does the step of ECL matter most? And how to adjust ECL reagent's ratio to
get better result.

Steps:
1. Harvest one 60mm dish of cells which have reached confluency by trypsiniz
ation. Wash it twice with PBS.
2. Add the cell pellet with 100 ul 1* sample buffer.
Boil it for 10 min
Pass it through a 21 gauge needle 10 times
Centrifuge for 10 min at 12.000 g, and transfer the supernatant to a fres
h tube
3. Run a 10% SDS-PAGE, 40 ul sample was loaded.
4. Transfer to a nitrocellulose filter
5. Block the filter with blocking buffer ( 2.5% nonfat milk in TBS)
Wash 3*5 min with TBS
6. Incubate the filer with primary Ab which is diluted in TBS.( for 90 min a
t RT)
Wash 3*5 min wih TBS
7 Incubate the filter with the secondary Ab which is diluted in TBS
Wash 3*5 min with TBS
8 ECL reagent
Reagent 1. 5 ml 100 mM Tris, PH 8.0
22 ul 90 mM P-coumaric acid
50 ul 250 mM Luminol
Reagent 2. 5 ml 100 mM Tris. PH 8.0
20 ul 30% H2O2

Autorad for 15 min

Are you running a positive control? if you are and you arent getting a signal then maybe the primary A.b. is not working. Are you getting high background? If you arent then the A.B. is suspicious. genereally i use the ECL from amersham and seems to work without a problem. 2ml of solution 1 and 2ml of solution 2. incubate for 1 minute and take a film. i would start looking at the A.b. if they are functinal then maybe its a lysate problem.

Try adding your antibody for a longer time. Primary over night at 4C and secondary for 2 hours at RT. Can also increase the concentration of primary and secondary antibody. I usually use 1:1000 and 1:5000 respectively.

Double check your protein concentration. Maybe you don't have enough target protein in your lysate for dectection. Lysis detergents can interfere with protein assay leading to an elevated protein concentration. Make sure your standards are prepared in lysis buffer and that they are linear.

I also would check the stablity or quality of your homemade ECL reagent. I've never made my own. Make up a batch, take 1 ml of it in a clear tube, go in the dark room, and spike it with 1 ul of 2ndary-HRP from the stock vial to see if it glows greenish. If it doesn't, either the solution or the anitbody is bad.

My signal usually develops with 10 second of exposure--2 minutes under the worst cases. Some ECL reagents have flash kinetics and then are stable for about an hour. If your working with 15 min exposures, your homemade ECL may also be decaying with each exposure requiring fresh batches be made.