Hi guys,
I did liposome flotation. Ithe liposome and protein were mixed in 30% sucrose(about 333ul), then overlaid with 200ul 25% sucrose, followed by adding 200ul HEPES buffer on top, then centrifugation(about 200,000g). However, I didn't find visible interface between 30% and 25% sucrose. I could only find the interface between HEPES buffer and 25% sucrose. Is it normal? How can I do to give 2 visisble interfaces? Thanks a lot!
Btw, I used TLA-100.4 rotor not Swinging Bucket rotor.
Feng
Several things you should check:
1) Is your protein stable in the sucrose solution?
2) If your protein is fine at room temperature then you can also do the spin at room temperature. Sometimes liposomes don't like the temp. change.
3) Did you test what the lowest sucrose percentage is that your liposomes can penetrate?
4) Sometimes membrane dyes can do strange things to the membrane. Did you check if your liposomes are ok (for example by EM)? Or has the size of the liposomes an influence on the binding of your protein?
5) If your floatation assay is giving problems, you may consider doing a pull-down experiment. In short, this means mixing your protein with liposomes, followed by a high speed centrifuge step to pellet those liposomes. If you analyse the pellet fraction and supernatant for the presence of your protein (using SDS-PAGE/Western) it should be possible to quantify the interaction.
You can find an example of such an experiment here: http://www.nature.com/nsmb/journal/v17/n3/full/nsmb.1770.html.
Method for floatation assay: (Reference: http://sci-hub.cc/10.1007/978-1-4939-3170-5_13)
Proteins are diluted with HP150 buffer to a concentration of 2 μM. 5 μL of 2μM protein is mixed with 45 μL of liposomes and incubated at room temperature for 10–30 min. 50 μL of the incubated protein–liposome sample is transferred into a 230 μL polycarbonate thick-walled tube and thoroughly mixed with 50 μL of 80 % (w/v) Nycodenz in HP150 by pipetting it up and down ten times. 50 μL of 30 % (w/v) Nycodenz in HP150 is then gently added on top without mixing.
Finally 30 μL HP150 is pipetted as top layer onto the Nycodenz gradient. The bottom layer is pink, whereas the 30 % Nycodenz and top HP150 layers are colorless. Place centrifugation tubes into adaptors for the S55-S swinging bucket rotor, which allows four samples to run at a time. Spin at 55,000 rpm (260,000 × g) for 90 min at 4 °C in a Sorvall Discovery M150 SE analytical ultracentrifuge.
Prepare six 1.5 mL Eppendorf tubes, each containing 15 μL 3× concentrated sample loading buffer. Carefully remove buckets from the rotor and take out the tubes. Liposomes form a pink rim on top of the gradient. Take six 30 μL fractions from the gradient and use a fresh tip for each pipetting step.
Heat Eppendorf tubes for 3 min at 95 °C. Analyze samples by Western blotting. The top two fractions of the gradient contain protein bound to liposomes and the bottom four fractions show the unbound protein.
I hope I could help you a bit!
Did you check your by EM PHOTO SCAN and transmission tbe interference must modulat protein concentration and posativlly correlated with sucrose
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