Cameron's LaBLog

 

Procedure: Purification
Have attached the 3mL column to position 7. Slightly more tubing than is ideal but this represents a reasonable "standard" setup. Dissolved a few mgs of GFP in water which means there will be some aggregate and injected 20 uL. Using the Tus buffer as eluent because there was some. At 0.3 mL/min pressure is around 1.2 MPa.

First run seems to indicate that tubing is an issue with peaks coming out later and much broader than in calibration run in brochure (not suprising as we have suboptimal injection, much more tubing etc) but also two peaks from GFP which seem too late for aggregate. So should have put the UV2 on at 490.

Second run, very little signal, probably a problem with injection, this could also be a significant issue with loading on small samples. Obviously much easier with proper injection systems. Suggests first peak is GFP second is solvent breakthrough ( no A490, plausible I guess but seems a strong response for water).

Third run, tried again, this time with the sample spun extensively. See if it shows a difference due to aggregation at all. Might need to heat sample or something to induce more aggregation to test separation and void volume perhaps? Seems to confirm that the first peak is GFP, second is solvent breakthrough. Maybe GFP is just a bit small as a test protein.

Changed to 100 uL loop to check effect of sample load volume. Given peak width suspect that it might not make very much difference. Now see a small aggregate peak at about 1.5 mL which now I know where it is is also in the earlier injection. After that things went badly wrong. Either have overloaded the column badly (but then why get good separation up to that point?) or have injected air onto the column.

After extensive washing tried again with a 100 uL load (53.6 mL). This time seemed to work well. Aggregate peak is separated from the protein peak with near baseline resolution. Resolution doesn't seem to be seriously impaired compared to the 20 uL load (peakwidth of the protein peak is comparable at around 0.8 mL in both cases).

Moved up to a 200uL load (73.7 mL). This is a new (and more dilute) sample. But resolution still seems to be good, a little worse at around (0.8-0.9 mL across the GFP peak, still good resolution from the aggregate).

Material: Solution
Project: smalp
SMALP sample B4
This Post is Linked By: SAXS on SMALP samples #1;

Procedure: SAXS
Instrument: I22
Project: smalp
following samples were run in 1.5 mm capillary cells on I22. Detector was at 5m and q range standardised against silver behenate.

Sample	Frames	Exposure time	I22 Raw Run #	Data
DLPC	30	10	29584	I22 29584-DLPC
A9	30	10	29585	I22 29585 - A9
A8	30	10	29586	I22 29586 A8
A7	30	10	29587	I22 29587 A7
A6	30	10	29588	I22 29588 - A6
A5	30	10	29589	I22 29589 - A5
A4	30	10	29590	I22 29590 - A4
buffer	30	10	29591	I22 29591 BUFFER

Data Type: SAXS_Diamond
Instrument: I22
Project: smalp

Radially averaged but not background subtracted

Raw data file	Data file
29591	J91001.DAT

This Post is Linked By: SAXS on SMALPS #2;

Data Type: SAXS_Diamond
Instrument: I22
Project: smalp

Radially averaged but not background subtracted

Raw data file	Data file
29590	J89001.DAT

This Post is Linked By: SAXS on SMALPS #2;