Wrong protein size on western blot - (Jun/05/2013 )

When I run my western blot I get very clear bands at 40kDa and very weak bands at 70kDa (Dnak). I have increased my blocking time from 1 to 2hours, but when I did this the bands at 70kDa could not be seen. I am not sure what the problem is? Could someone pls help!

Can you give more details of your method, for us to work on? There are many potential reasons why you might not be seeing the appropriate bands.

I grow the bacteria to mid-log phase, expose to stress(ethanol, heat shock, and antibiotics) for 1 hour and 4 hours. Take 1ml aliquots and centrifuge. I resuspend the cells in 50 microliter sample buffer (1x) (4x containing 0.5M Tris-Hcl, glycerol, 10%SDS, Beta-mercaptoethanol, 0.05% and about 1 gram DTT). Then I run 20microliter of the samples at 90 volts for 2 hours. I transfer at 250mA for an hour using nitrocellulose membrane/PVDF. Then I block the membrane for an hour/two at rt, incubate in primary antibody overnight at 4degrees, incubate in secondary antibody for 90 mins-120 mins. I expose for 24 hours as the signal is very weak.

OK - most of that looks OK. Incidentally you shouldn't need both DTT and B-ME in your buffer as they both do the same thing.

What are the compositions of your blocking and antibody incubation buffers?

is your protein a single or multiple subunit?

is 70kDa the subunit or native size?

if your answer to both is the latter choice of each question then what you are seeing is probably correct.

5% low fat milk in TBS/tween (0.2%). I do a 1:5000 dilution of both the primary and secondary antibody. I did start off with 1:10000 but the signal was very weak.

It's a monomer and that is the native size but it does bind to two co-chaperones to properly work. Is it possible one of the co-chaperones are being picked up? Is that possible cause I am using a primary monoclonal antibody against DnaK?

you increased blocking time on a new blot from a new gel?

you may be experiencing proteolysis of your sample stock.

can you try an fresh preparation?

Yea, I used a new blot and new gel. And I never reuse my samples. I always make fresh samples for the western blot.

So you havn't titrated the antibody at all or is this standard procedure for this protein in your lab? It is unusual (but not unknown) to find a primary antibody that will work well at 1:5000, most work well in the 1:500-1:2000 range. Try titrating the primary - you may well find that your 70 kDa band is enhanced. You can also lower the detergent content in the TBS-T, usually 0.05-0.1%.