For western blots, I normally stain with 10 mL buffer with 1:1,000 dilution of my specific antibody.
Recently, there is an equipment change in my lab and 10 mL isn't enough volume to cover the membrane. If I want to use 20 mL of buffer, should I change the antibody dilution? Should I use 1:2,000 so there is still the same total amount of antibody? Or should I use the same dilution?
It's better if you maintain the dilution factor as you used before 1:1000.
keep the dilution factor provided you have no issues with running low on antibody (as it is a lot of antibody). If the volume of antibody is a concern in that you will eat through your stocks more rapidly, you could try and lower the dilution as there is a chance 1:2000 can work just as well as 1:1000 (this is something you would have to test/optimize if you haven't already).
Your worry is usually that you soak up all the available antibody and can't generate a strong signal or get a consistent blot. You would probably be fine to not increase the amount of antibody, but then you need to consider that your binding might decrease depending on how long you incubate for since the antibody is more dilute and may not see the epitopes as readily. You would need to test/optimize as already suggested to see if there is any affect and maybe try incubating for longer if there is.
Alternatively, you can look into heat sealing the membrane in a plastic bag (I use plastic sheet protectors) which can reduce your volumes to 0.5ml-2ml depending on how large the area you want to blot and then put on a shaker with flat weight on top (think like a heating block). This saves a lot of antibody. There is another method of floating your blot using parafilm to put antibody/buffer mix and laying your membrane on it (no agitation and be careful of evaporation so place it in a sealed box/dish with wet kimwipes or some other method to make a humidified chamber).
What is the size of your membrane? I usually use 3mL of buffer diluted with specific antibody to cover the membrane (size: 8.5cm × 6cm) without any problem. Its better to follow recommended dilution factor of specific antibody.
Given that 1:1000 is the recommended dilution of the antibody of your interest for the western blotting, better you maintain the same dilution factor for a better signal.
Moreover, there is no recommended equipment as such for staining the membrane. One can use any holder that can accommodate both the membrane and the antibody solution taking care that the membrane is completely immersed in the Ab sol. if you can accommodate the membrane, you can even use a 50ml tube with 5 ml of Ab sol and leave it on a roller. this will even save 5ul of valuable Ab. If the membrane is small, one can even miniature the entire staining process.
But it is always recommended to maintain the antibody concentration to have a better signal.
- The equilibrium constant of antibody-binding is defined by the concentration, not the amount, therefore you should keep the concentration constant.
- What is the size of your membranes, that they require 20 ml of AB?
- Which antibody are we talking about: primary or secondary?
- In my experience, the primary antibody solution in TBS-T with a suitable preservative (sodium azide or mersalyl) and 1% BSA keeps for years in the fridge and can be used many times if the incubation is done at 4 °C o/n (for this solution I do use BSA instead of the normally preferred milk powder, as the long-term stability of milk powder is lower).
- In case of the secondary antibody, which is usually discarded after one use to avoid loss of activity of the coupled enzyme, the price is relatively low, so that doubling the amount used per test contributes little to the total costs of the test. Nevertheless, I'd check whether the large volume is really necessary.
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