Example of Western Blot using Global Antibodies
Electrophoresis and Transfer Equipment:
- Electrophoresis apparatus:InVitrogen XCell.
- Gels and buffers: InVitrogen NUPAGE BisTris Gels with
either MOPS or MES as running buffers (gives different
separation ranges).
- Transfer membrane: PVDF (other membranes will work as well)
Transfer Conditions:
- 10 % methanol
- Transfer time: 60 min in 1X NuPAGE Transfer buffer.
For 2 gels, additional time ( up to 80-90 minutes in total) improves transfer of larger proteins.
Method:
Blocking -TBST (pH 7,6 + 0,05% Tween20 + 5 % nonfat dry milk.
1 hour at room temperature or overnight in the cold room while shaken.
No wash necessary when going from block to primary antibody incubation
Primary antibody incubation.
Primary antibody added to TBST with 2% milk.Incubation done 1 hour at room temp on shaker. Global
antibody dilution from 1: 2000 to 1: 10 000, depending upon tested material.
Washes.
One quick rinse, followed by two 10 minutes washes and one 5 minutes wash, all done with TBST.
Secondary antibody incubation.
With global antibodies secondary antibodies from following companies has been tested:
Abcam, Pierce, Sigma.
Secondary antibody from Abcam HRP conjugated at 1: 6 000 in TBST with 2% milk incubated for 45
minutes at room temperature.
Washes.
One quick rinse, followed by two 15 minutes washes and one 5 min wash, all with TBST.
During the washes prepare ECL developing reagent.
Reaction develpment.
Pour off final wash.Pipette ECL mix onto blots. Wait 1 minute. Drain off developing reagent.
Cover the membrane with of transparent wrap (Saran works best).
Expose to film or Fluorescence detection system.
Notes.
<• So far, in our hands, best results with global antibodies have
been obtained using ECL Advance system from Amersham Biosciences in conjunction with the NuPage electrophoresis and blotting system from Invitrogen.
High sensitivity of the reagents enables use of global antibodies at high dilutions.li>
- Using different secondary antibodies as well as detection systems can
actually give different results, due to other sensitivity of chosen secondary antibody-detection
reagent pair.
- Any solution containing milk should be made at least 15 min prior and stirred to dissolve
well.
- TBST made by pipetting 0.5g of Tween 20 (very viscous liquid) into small beaker,
adding 1X TBS, and stirring to take Tween20 into the solution.
- Trays used for incubations should have absolutely flat bottom. For overnight blocking complete
coverage of the membrane with solution should be assured.