1. Is it okay to use this plate?

24-well if you have now idea and use normal western? No. Take a 6-well. I mean A549 are not difficult to grow Or precious

2. What cell confluence is needed to conduct this experiment?

well, Hypoxia is stress for cells and can influence their behavior (See HIF-2a and autophagy). Try 70-80% density if you want to stimulate autophagy just by your stimulation.

3. How much lysis buffer should I use?

depends. Start with 50microliter

4. I was thinking of having 2 controls and 2 experimental wells for each concentration, should I put the contents of each well in a separate centrifuge tube? Before you start worry about your experiment, you have to titrate your amount of lysate to figure out how much total protein you need. Best you titrate you4 ab as well. Pool some wells to compare enough samples.

5. Does the LC3 antibody bind both LC3-II and LC3-I?

depends on your ab. Read the manual.

6. How much of the lysate samples should I load into the wells of the gel? One protocol suggested between 10 and 30 μg.

see above. It depends on how the a549 express your POI

7. What voltage should I use for the protein electrophoresis and for how long?

depends on gel, size of protein...

8. What voltage should I use for the membrane transfer and for how long?

depends on gel, size o& protein...

mh, if you have never done a western, maybe start with the loading control. It might get difficult if you have no clue about wha5 you are doing. And get some proper help. You might need someon3 who is teaching you first. Sorry, but otherwise you might waste a lot of time and money, my dear

best,

Teresa

Dear Teresa,

Thank you for the help! I will have someone teaching me but I thought I would get some help online first. I will keep all your answers in mind. Thanks again.

regards,

Gjon

If you have never done PAGE and Westerns before, perhaps do those with a cheap sample (like serum) first. Stain the gel either with permanent stains like RuBS (doi:10.1007/978-1-59745-542-8\_14) and/or with the non-permanent Zn/imidazole (doi:10.1016/j.ab.2004.02.017). The blots you can stain with India ink (doi:10.1016/0003-2697(83)90237-3) or with Ponceau S (doi:10.1016/0003-2697(86)90263-0).

Once you can perform these basic techniques, you can try to make cytosol preps from your cell line. Note how much protein you get from a given number of cells. For WB, 30 µg of protein for a well in a mini gel is a reasonable starting point, unless your protein of interest has very low copy numbers.

For electrophoresis, constant current (10 mA per gel while the front is in the stacking gel, 20 mA during separating gel) is about right for a mini-gel (6 cm x 10 cm), hardly anybody is using the large 20 cm x 20 cm gel anymore. Constant current is preferable over constant voltage because the conductivity of gels is much higher at the start than later on. In constant current mode, the gel always gets as much power as it can handle without too much heat generation.

For blotting, there are two systems,

• semi-dry where the source of buffer are sponges on both sides of the gel/membrane pack, the assembly is pressed between a pair of carbon electrodes
• tank blotters, where the gel sandwich is immersed in a tank with buffer.

The latter IMHO is clearly preferable, probably because of better heat dissipation. The semi-dry systems you need only if you want different buffers on gel and membrane side (doi:10.1006/abio.1993.1313), or if your protein of interest is very small (< 10 kDa or so). 30 V for an hour is a good starting point, stain the gel afterwards to verify complete transfer.

Use PVDF membranes for blotting, not nitrocellulose. Protein binding affinity and capacity are way better, and they do not fragment during the many incubation steps with antibody.

Many people use Towbin's buffer for blotting (doi:10.1073/pnas.76.9.4350), but Dunn's works better and is a lot cheaper (doi:10.1016/0003-2697(86)90207-1).

Last but not least two advises on safety:

• Acrylamide is a potent neurotoxin that can cross intact skin. Handle with due care, in particular with gloves. Polyacrylamide is harmless (used to make contact lenses), but may contain unpolymerised acrylamide until thoroughly washed (which happens during fixing and staining). Handling gels with gloves also prevents finger prints that stain with sensitive methods like RuBS or silver.
• High voltages and buffers with high conductivity are a "ticklish" mixture! Follow the user manual and always keep one hand in the pocket of your trouser.

Dear Engelbert,

Thank you for the detailed response. It is of great help!