Wasting Antibodies Doesn’t Float Your Boat? Try Floating Your Blot Instead!

Western blots may be great for visualizing protein expression, but they can be a perfect way to waste your precious antibody stocks if you follow the normal protocol. Thankfully, you don’t have to follow the normal protocol any more; here’s how to get great blots with a fraction of the antibody usage.

 I have tried several different techniques to minimize the amount of primary antibody that I use. In some labs, I have used heat-sealed bags to create a bag that is just slightly bigger than the blot. This can significantly reduce the amount of liquid required to cover the blot.

 More recently, without access to a heat-sealer I turned to diluting my antibody in the smallest volume of dilution buffer possible to just cover a blot laying in a Tupperware container that is slightly larger than the blot (usually 7-10mls for a 9×5.5 cm membrane). Call me frugal, but even this approach still makes me cringe for antibodies that are diluted only 1:500-1:1000.

Floating your blot

But my newest (and now favorite approach) is one I call “floating your blot”. It requires a Tupperware container with a sealable lid and a flat bottom, parafilm, kimwipes, forceps, antibody dilution buffer and primary antibody. A 9×5.5cm membrane requires only 1mL of antibody diluted into buffer. To float your blot, follow these easy steps:
 1) Cut a piece of parafilm that is slightly larger than the membrane.
 2) Pipet some antibody dilution buffer without antibody (I usually use 1% low-fat dry milk, 0.1% Tween, PBS) onto the bottom of the Tupperware container and spread around using a kimwipe.
 3) Lay the parafilm onto the wetted area without creating bubbles between the parafilm and the container. This sticks the parafilm to the bottom of the container.
 4) Wipe off excess dilution buffer
 5) Dilute antibody in antibody dilution buffer. I use 1mL for a full membrane (9×5.5 cm), and 0.5mL for half a membrane.
 6) Pipette diluted antibody onto the parafilm near one end.
 7) Using forceps, pick up membrane that has been incubated in blocking buffer and lay protein side down onto the diluted antibody. I lay one edge of the membrane against the liquid and allow the membrane to wick across. Keep the membrane on the parfilm and be careful to prevent any bubbles from forming between the liquid and the membrane.
 8) Seal container with lid. This must be an air-tight seal or the diluted antibody will evaporate.
 9) Incubate with primary antibody using conditions optimized for the antibody.
 10) Remove blot into new container for washes.
 11) Technique can be repeated for incubation with secondary antibody if desired. I wish I could take the credit for thinking of this on my own, but I didn’t. A colleague showed me this trick and it really helps me save my antibodies.

Author:

Rebecca Tirabassi, BitesizeBio

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How To Read Manuscripts (and anything else) Twice As Fast

I always thought that doing a speed-reading course would be a good thing to do as a scientist. With the amount of literature we need to consume, speed-reading (the art of reading faster without reducing comprehension) would save a lot of time.

But it turns out that you don’t really need to spend any cash on a course to teach you to read more quickly. There are some pretty simple steps that you can use to dramatically improve your reading speed that won’t cost you a penny.

The basic principles are:

1. Use a tracer, like a pen, or your finger, to trace under each line as you read it, as if you were underlining. You do not read text in a continuous line, but in a sequence of jumps, each of which ends with a temporary snapshot of the text within you focus area. Subconsciously, you will spend a lot of time re-reading and skipping back to previous snapshots. Using a tracer helps your eye focus and prevent this from happening.

2. Train yourself to use your peripheral vision as you read. When we read, we tend to focus just on the words that are in our central focus in each snapshot. But you can train your eye to use your peripheral vision too and so multiply your reading power.

10 minute speed-reading training
The following 10 minute training routine is based on these principles. I’ve tried it a few times recently and have seen a notable improvement in my reading speed:

1. For 2 minutes: Practice reading as fast as possible, using a tracer to guide you. Don’t worry about comprehension to begin with – that will come.

2. For 3 minutes: Train your eye to use your peripheral vision by focussing on the THIRD word and the the THIRD FROM LAST word in each line.

e.g. if the line was this, you would focus on the underlined words:

“We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.).”

As you practice this and it becomes easier, you can start to move your focus into the fourth and fourth from last words and so on.

3. For 2 minutes: Practice reading each line in only two snapshots using your focus on the third and third from last words as an anchor.

4. For 3 minutes: Read too fast. Using techniques 1-3, practice reading too fast for comprehension for a couple of pages and then (still using the techniques) slow down to a pace that you can comprehend the text. This will help accustom your brain to reading more quickly — a bit like when driving a city speeds seems very slow when you have just come off the motorway.

Obviously the more you repeat the cycle, the faster you will get. If you have a go, please drop me a comment – I’d be interested to hear how it went for you.

ps: If you want to monitor your progress, measure your reading speed (words per minute) before and after. You can calculate the average number of words on a page by calculating the average number of words in 10 lines, then using this to calculate the average number of words per page and so on (but you don’t need me to tell you that!

by Nick Oswald in Organisation & Productivity
From BiteSizeBio

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How a Jellyfish Changed Biology: the discovery and development of GFP

Fluorescent tags are widely used for microscopy and expression studies – but it wasn’t so long ago that this everyday tool was unheard of. In this article we’ll talk about how GFP came to be, and what it means for you.

Green fluoresecent protein, or GFP, was first identified in a fluorescent jellyfish, Aequorea victoria. Osamu Shimomura purified GFP and described the biophysics of how it fluoresces. A few years later, Martin Chalfie reported the expression of this protein in E. coli and C. elegans. Roger Tsien is responsible for designing variants on the protein – single amino acid changes that yielded cyan, blue and yellow fluorescent proteins, and the enchanced green protein (EGFP) that is commonly used today. Shimomura, Chalfie and Tsien were awarded the Nobel Prize for Chemistry in 2008 for their work in the discovery and development of this tool. What made GFP such a game changer is the fact that it’s “auto-catalytic” – it doesn’t need any co-factors or enzyme processing to fluoresce – so it can be easily used in a wide variety of organisms.

The major applications for GFP proteins are microscopy based, since its primary value is as a visual marker for protein detection. Here are a few of the most popular ways to use GFP:

1. Translational fusion
One of the most common uses is a fusion marker, where the GFP open reading frame is cloned downstream of your favorite ORF, so that it is translated as one long protein, fusing your favorite protein to GFP. That way, wherever your protein is expressed you will see green fluoresence. This can be used in still images and is striking in images of live cells, as you can track the location and movement of proteins.

2. Transcriptional fusion
GFP can also be used in “transcriptional fusion”, where the expression of a gene and GFP are driven off the same promoter, but with an intervening stop codon. In this case, cells expressing the first gene will fill with soluble GFP – resulting in easy detection of the particular cells expressing your protein.

3. FLIP and FRAP
FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) rely on the fact that a single GFP molecule emits fluorescent light when it’s excited, but cannot do so indefinitely. Eventually it either bleaches out or stops emitting. So, to study the dynamics of a GFP-labeled protein, you can bleach a small area of a cell and determine how long it takes fluorescently labeled protein to “leak” back into the bleached area (FRAP), or how much fluorescence decreases overall in the rest of the cell as the bleached proteins diffuse (FLIP).

4. FRET
FRET (fluorescence resonance energy transfer) is based on the different excitation and emission spectra of the different variations on GFP. In this case, two proteins are labeled with two different fluorophores, which are carefully selected so the emission spectrum of the first overlaps the excitation spectrum of the second. The cells are then imaged using a laser that excites only the first fluorophore – so the second only lights up if the two proteins are in close enough proximity that the first fluorophore sets off the second.

Check out some of the seminal papers written about GFP:
Green fluorescent protein as a marker for gene expression.
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC.
Science. 1994 Feb 11;263(5148):802-5.

Wavelength mutations and posttranslational autoxidation of green fluorescent protein.
Heim R, Prasher DC, Tsien RY.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12501-4.



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