Ultrasonic bath for ChIP antibody incubation - (Jul/10/2009 )
I have an R&D Exactachip kit but this is also relevant for the Fast ChIP protocols (such as: http://knol.google.com/k/jonathan-jacobs/f...51cfalk93s5s/3# ).
I am wondering if there are any specifications regarding what type of ultrasonic bath to use for the 15 minute antibody incubation step.
I ask this for a particular reason because in our lab we have an Ultrawave U100H ultrasonic bath (specifications below). However a group in our lab uses this bath to actually shear the crosslinked chromatin for ChIP. They simply place the samples (within 1% SDS lysis buffer) in a thin floaty into the ice and water filled (approx 2 degrees celsius) bath and sonicate for 5 mins. They then use this sheared chromatin for ChIP using the traditional lengthier antibody incubation method at 4 degrees.
Therefore it seems likely that it would not be possible to use this ultrasonic bath for incubating my antibody in Fast ChIP protocols because my already sheared chromatin (sheared by whatever method) will be further cut. However having read almost every thread on this forum regarding sonication the only info I can discover is that an ultrasonic waterbath should not be powerful enough to shear chromatin.
So essentially I'm a bit confused, and wondering if there are more and less powerful ultrasonic baths and whether the one we have is too powerful. (Unfortunately I can't seem to find much information about the specifications of the Branson 3510 ultrasonic bath used in the protocol at the top.)
Any thoughts would be much appreciated.
Ultrawave U100H
Bench Top Ultrasonic Cleaning Bath, 1.5Litres Working Capacity Analogue
Tank dimensions are 150mm x 140mm x 100mm Deep.
Specification
Construction Case Rigid Stainless Steel
Construction Tank Stainless Steel
Operating Voltage 240 Vac 1ph 50Hz
Power Consumption Ultra 65 Va Rms
Power Consumption Heater N/A
Transducer Bonded 1 - 50mm Ceramic Disc
Power Output P-P Watts 130 Watts
Timer Range 0 - 15 Mins
Tank Dimensions 150 x 140 x 100 Deep
Max Capacity 1.75 Litres
Working Capacity 1.5 Litres
Operating Frequency 30 - 40 Khz
Modulation 50 - 100 Hz
Overall Dimensions 180 x 160 x 190 Deep
Net Weight 3.2 Kgs
Gross Weight For Export With Lid, Basket & Packing 4.0 Kgs
mattbarter on Jul 10 2009, 04:20 AM said:
I am wondering if there are any specifications regarding what type of ultrasonic bath to use for the 15 minute antibody incubation step.
I ask this for a particular reason because in our lab we have an Ultrawave U100H ultrasonic bath (specifications below). However a group in our lab uses this bath to actually shear the crosslinked chromatin for ChIP. They simply place the samples (within 1% SDS lysis buffer) in a thin floaty into the ice and water filled (approx 2 degrees celsius) bath and sonicate for 5 mins. They then use this sheared chromatin for ChIP using the traditional lengthier antibody incubation method at 4 degrees.
Therefore it seems likely that it would not be possible to use this ultrasonic bath for incubating my antibody in Fast ChIP protocols because my already sheared chromatin (sheared by whatever method) will be further cut. However having read almost every thread on this forum regarding sonication the only info I can discover is that an ultrasonic waterbath should not be powerful enough to shear chromatin.
So essentially I'm a bit confused, and wondering if there are more and less powerful ultrasonic baths and whether the one we have is too powerful. (Unfortunately I can't seem to find much information about the specifications of the Branson 3510 ultrasonic bath used in the protocol at the top.)
Any thoughts would be much appreciated.
Ultrawave U100H
Bench Top Ultrasonic Cleaning Bath, 1.5Litres Working Capacity Analogue
Tank dimensions are 150mm x 140mm x 100mm Deep.
Specification
Construction Case Rigid Stainless Steel
Construction Tank Stainless Steel
Operating Voltage 240 Vac 1ph 50Hz
Power Consumption Ultra 65 Va Rms
Power Consumption Heater N/A
Transducer Bonded 1 - 50mm Ceramic Disc
Power Output P-P Watts 130 Watts
Timer Range 0 - 15 Mins
Tank Dimensions 150 x 140 x 100 Deep
Max Capacity 1.75 Litres
Working Capacity 1.5 Litres
Operating Frequency 30 - 40 Khz
Modulation 50 - 100 Hz
Overall Dimensions 180 x 160 x 190 Deep
Net Weight 3.2 Kgs
Gross Weight For Export With Lid, Basket & Packing 4.0 Kgs
Hi,
Yes, the ultrasonic bath in your lab is too powerful for the antibody/chromatin interaction. When we developed Fast ChIP we used two different ultrasonic baths, one which was older than dirt and I couldn't read the manufacturer and a Branson 3510. It doesn't seem like the specific brand doesn't matter that much, just that power level isn't too high. I've corresponded with one person who used an ultrasonic bath used for cleaning jewelry and it worked fine for him.
Good luck,
Joel
Thanks for the quick reply!
Is it the 50-100 Hz modulation which signifies that it's too high?
mattbarter on Jul 14 2009, 09:15 AM said:
Is it the 50-100 Hz modulation which signifies that it's too high?
I don't think it's the frequency that's the problem but more likely the power output. Unfortunately, most ultrasonic baths (the ones you would use for the incubation with the antibody) don't have control over the power output, which is factory set. A colleague of ours bought one of the Bransons (same model as what we use) but the power output was so high it was deforming the tubes (the ChIP didn't work either so the power output is definitely and issue).