Mail-In Instructions for SAXS data collection at SIBYLS
Sample Format
Sample Quantity - 24µL above 1mg/ml
Buffer properties - Do not use greater than 10% glycerol, it has been reported that 2% is the maximum benefit. With glycerol the viscosity of the solution increases and bubbles become more of a problem.
Please order a plate and cover from
http://vwrlabshop.com/96-well-pcrand%23134%3B-plates-axygen-scientific/p/0022804/
1. Full skirt 96 well clear plate VWR catalog # 10011-228
Pick a colors to aid in uniquely identifying your plate.
- AxyMat Sealing Mat VWR catalog # 10011-130
Surface tension is pretty high in these plates but the drops will fall out if the plate is dropped on a hard floor. We still recommend over night shipping "rubberbanded" between 4°C ice packs after purification, but flash freezing should also be possible. Slow freezing is nearly the worst thing you can do to your protein and will happen if you put your liquid samples directly on an ice pack previously stored in the -20°C. The more 4°C in the box the better.
Please write your labs name on the side of the plate.
Fill up the plate with 24ul of the sample and buffer as follows:
A1- buffer 1
A2- protein 1 (low concentration)
A3- protein 1 (medium concentration)
A4- protein 1 (high concentration)
A5- buffer 1
A6- buffer 2
A7- protein 2 (low concentration)
A8- protein 2 (medium concentration)
A9- protein 2 (high concentration)
A10-buffer 2
A11 ...
A12 ...
More information below in spreadsheet section.
Please send to:
Lawrence Berkeley Lab
1 Cyclotron Road
MS 6R2100
Berkeley, CA 94720
ATTN: Jane Tanamachi / 12.3.1
510-495-2404
Please send filled out spread sheets to Kevin, Jane and Greg along with a tracking number for your samples.
Required Information About Samples - Spreadsheet
Please alter the attached spreadsheet for your experiment. Download Spreadsheet
First column is the well number - do not change any value in this column
Second column indicates the order in which the contents of wells will be collected
Third column has an "x" for samples after which a wash of the sample cell is preformed. Limit washing a
s much as possible since this is the primary bottleneck. In the example spreadsheet, note that washing is only done between the highest concentration and the buffer. Even when switching buffers washing is not always necessary as we wash with water and this is probably a greater difference than between your 2 types of buffers.
Fourth column has an "x" if the sample in this well is a buffer. This will automatically put a "buff_" prefix on the file name.
Fifth column is the name of the data output file.
We will ignore information in the other columns - they are a work in progress.
Information about Example Experiment in spreadsheet
The attached spreadsheet has a template experiment loaded where 5 proteins are being studied. SAXS is a difference technique so an exact buffer blank is required with each sample. In the template each protein is studied at three concentrations - one third, two thirds and full concentration. The lowest concentration we can reliably attain data for is 1mg/ml. Note the low to high order of concentrations and a wash step only after the highest concentration was collected. The first three proteins were all in the same buffer and buffers were collected before and after each sample. The remaining 2 proteins were in unique buffers requiring extra buffer collections and wash steps between buffers. Please follow this template with your samples.
What Will Happen When Your Sample Arrives
You will be notified that the samples have arrived.
The samples will be placed in the 4°C fridge.
The plate will be spun at 3000 rpm for 10 min at 4°C prior each data collection.
The plate will be at 10°C during the data collection. Each sample will be loaded into the SAXS cuvette which is at 20°C for 15 seconds.
Data will be collected as soon as possible by SIBYLS staff and you will be notified when data is available.
What You Will Get Back
You will get back 3 integrated scattering profiles in ASCI for each sample where the 3 are different exposures 0.5, 1 and 6 seconds.
You will get a spreadsheet which displays your scattering profiles and an initial assessment of the sample from beamline staff.
More detailed information about the results will also be sent with samples.
More Information
We have a great deal of information on the web at
http://bl1231.als.lbl.gov/forum
We welcome questions and constructive comments and will do our best to address both.