Question from Stephanie about Tween 20

Stephanie is a lab technician who is getting ready to use a Western Transfer detection procedure to see where in the world her protein lies on her Western membrane.

Stephanie states that her advisor got an affinity purified antibody, to the protein that he's studying from a colleague in Connecticut, and he handed the antibody vial along with an "ECL Western Blotting Detection Reagents and Analysis System" Kit from Amersham Biosciences.

When she read the instructions she discovered (to her shock, horror and dismay) that she needed a substance called Tween 20 and...

When she looked in the kit no such item was present.

Turns out that Tween 20 and water need to be supplied by the customer.

Who would have known.

So now she needs to use something that she's never heard about...

Here's her question, in her exact words:

"What is Tween 20 and where can I get some for my detection procedure?"

This is not the type of question that pops up at your average family get-together. But if it did this is how you could answer.

Oh, and don't forget that if you're a guy and you are communicating to a gal remember that they like to hear nice things about themselves. The more nice things you can say the better but don't dump a ton of nice things all at once because they'll suspect somethings up so watch and learn how I skillfully handle this delicate balancing act in my response.

"Aaahhh Stephanie... I'm so glad you asked. Oh, by the way...

"I like your earings... very complementary. Now, about Tween 20.

"That is a registered brand name for a chemical that is found in some shampoos or soaps.

"The chemical name is polyoxyethylene-20-sorbitan monolaurate.

"The molecule consists of 58 carbon atoms, 114 hydrogen atoms and 26 oxygen atoms.

"It's formula weight is 1,227.54 grams per mole.

"Ooh, and did I compliment you on your nails? Nice choice of colors.

"Since you mentioned that you need some for a detection procedure, and since I heard through the grape vine that you're working for Dr. Zippity-Doo-Dappity-Ding-Dong (who happens to be a big shot in the field of protein chemistry) I'll assume that you need the Tween 20 as a Western blotting detection reagent component.

"I see that your nodding yes. That's good... ooh, nice eye-shadow... applied just right... not too thick, not too thin... just right...

"I recommend enzyme grade Tween 20 and you can get some from FisherBiotech. I believe a 500 ml bottle (dark brown, transparent glass) sports the following product number: BP337-500.

"Since this is a concentrated soapy material don't get any on your skin, in your eyes or in your digestive tract.

"And, as always, wear protective eye guards when working in the lab. You certainly wouldn't want to hurt those gorgeous peepers of yours.

"Oh, did I say peepers? I meant your 'photon collectors'. They are very nice."

Disclaimer: Neither Stephanie or the answer-person is being paid or remunerated in any way by FisherBiotech or Amersham Biosciences, its assigns or agents. Information provided in this missive is purely for educational and entertainment purposes and the author is not attempting to provide legal, medical, accounting or psychological services. The author is not responsible for any damage or harm that may occur to any reader due to the actions of those readers that may or may not have been catalyzed by the words in this message.

From West-Gate Press and sell info products online dot com (sellinfoproductsonline.com) and basic drum beats dot com (BasicDrumBeats.com) and wokatoosie dot com (www.wokatoosie.com) this is LeBlond, Gerard LeBlond and I'll see you there..... Cheers.

Organize Your Lab For Top Efficiency

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Hi:

This is LeBlond, Gerard LeBlond and I was asked the following question yesterday:

"We've got pipettors strewn all over the lab and a lot of time is wasted looking for the 1 to 10 microliter dispensing one from the 2 to 20 microliter dispensing pipettor. Also...

"Our refrigerator and freezer common areas are cluttered and disorganized. Any suggestions for bringing order to our lives?"

Great questions...

You need pipettor racks to organize your liquid dispensing devices.

Acrylic racks can be purchased that fit either on the surface of your bench or that can be attached to the front edge of a shelf.

If you've got 3 work areas and have enough pipettors to outfit each of those work spaces get a rack for each location and remember to return a pipettor to it's proper storage space soon after use.

How soon?

Right after you use it you put it back in its holder... "That's how soon!"

Refrigerator and freezer reagent inventory storage systems are commercially available and some are even "lockable".

There are a number of suppliers that can fill your lab's "organization needs" like Fisher Scientific, VWR and Becton, Dickinson and Company.

Once source of "hard to find lab" equipment can be found on the web at mlMarketLab.

Good luck and go out and clean up that lab...

Stat!

Flagellar Researcher Dr. Elizabeth Smith Seminar Announcement

On Tuesday 26 October 2004, at 12 Noon, in room 103 of Weiskotten Hall of SUNY UMU in Syracuse, NY there will be a seminar entitled: "Calcium Regulation of Flagellar Dynein Activity: A Role For Multiple Calmodulin Associated Complexes Anchored To The Axoneme".

The invited speaker is Dr. Elizabeth Smith, Associate Professor in the Department of Biology of Dartmouth College.

She will be the guest of Dr. David R. Mitchell of the Department of Cell and Developmental Biology at SUNY UMU.

Admission is free!

Top Two Books For Flagella Researchers

LeBlond, Gerard LeBlond, Ph.D. from SUNY UMU in Central New York State.

I recently received the following question: "I'm thinking about branching out in my research activities and heard that Chlamydomonas reinhardtii is a great model organism. What are the two reference books that you suggest for doing research on this beast?"

Here's my short list:

• Methods in Cell Biology Volume 47: Cilia and Flagella, Edited by William Dentler and George Witman. Academic Press, 1995 Hardcover: ISBN 0-12-564148-6; Softcover: ISBN 0-12-209710-6
  
• The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use by Elizabeth H. Harris. Academic Press, 1989 ISBN 0-12-326880-X
  

Good luck finding your own copies.

But if you do get your own tomes for your library -- Hold on to them -- because...

They are fantastic!

Technique To Affinity Purify Antibodies From Immunized Animal Sera Part 2

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Hello:

This is LeBlond, Gerard LeBlond from Central New York State.

This is Part 2 of a series of posts concerning how to Affinity Purify Antibodies From Immunized Animal Sera.

Before we get into the specific materials that you'll need, prior to starting the procedure, let me reveal the basic technique.

The procedure needs, and centers around, an hydrophobic membrane that has your protein of interest bound to it.

This membrane would be prepared using "Western transfer" techniques involving a preparative or curtain-type SDS-Polyacrylamide Gel.

The preparative (curtain-type) sodium-dodecyl sulfate-polyacrylamide gel (SDS-PAGE gel) is cast (prepared) using a very wide comb so that the "loading well" can accommodate a large volume of your sample (i.e. protein in solution with loading dye).

The comb I like to use lets me simultaneously create one large preparative well and a small well.

The former is used to load the solution containing the sample of interest (sample) and the latter is reserved for loading a solution of protein size markers which are important for "size calibration" purposes.

So here's the procedure in condensed form:

• cut the Western Membrane to yield a strip containing your bound protein of interest (membrane A) and two other pieces that contain proteins that are other than the protein of interest (membrane pieces B);
  
• Block the membranes A and B;
  
• Expose anti-sera to membranes B to "scarf-up" (deplete; scavange; sequester) antibodies that do not recognize your protein of interest;
  
• Transfer anti-sera from tube containing membranes B to membrane A to allow the antibodies that recognize your protein of interest to bind to those proteins on membrane A;
  
• Wash membrane A;
  
• Treat membrane A to an alkali soak to release any antibodies that bind to your "protein of interest" that may dissociate (unbind) at high pH;
  
• Collect the alkaline soak solution
  
• Neutralize membrane A and the "alkaline soak" solution;
  
• Treat membrane A to an acid soak to dissociate antibodies that bind to your "protein of interest" at low pH;
  
• Neutralize membrane A and the "acid soak" solution;
  
• Pool your neutralized "alkaline soak" and "acid soak" solutions and dialyze against a buffered salt solution;
  
• Add a preservative (antifungal and antibacterial) to your dialyzed solution and store the solution (enriched in your antibody of interest) at either 4 degrees celsius or -20 degrees celsius.
  

We perform both an alkaline and an acid "soak" because we don't know if the-antibody-that-we-are-interested-in dissociates from our "protein of interest" at high or low pH.

Consequently we do both procedures and pool the neutralized solutions to be on the safe side.

If we knew that our antibody disssociated with the acid soak then we could naturally skip the alkaline soak, and vice versa. But...

This information isn't normally available to us unless we specifically test to find out.

You might ask, "I want to find out!" And...

I'll reply, "Well, why didn't you say so before!"

If you want to find out if the antibody dissociates from "your particular protein of interest" just do not pool the neutralized alkali and acid pools... conduct separate dialyses of each batch and test to see which batch (alkaline or acid) contains your affinity purified antibody.

O.K. Next...

Here are the materials that you will need to perform this procedure:

• Western membrane (PVDF = polyvinylidene difluoride = hydrophobic support) containing your protein of interest;
  
• Anti-sera from animals immunized with your "protein of interest" using standard operating procedures, and commonly accepted practices, for conducting such a procedure (we out-source this procedure... apologies to "Union Guys and Babes" who insist, in their labor contracts, to keep all things 'in-house', but...
  
  If you can out-source a procedure like this you'll save yourself a lot of headaches but...
  
  That's assuming you commission a reliable/established/efficient lab with a proven track record for successfully completing such procedures on schedule and on-budget...
  
  Otherwise take your chances with the "union guys" or do it yourself. But...
  
  Don't tell me I didn't warn you!); you'll also need:
  
  
• Scissors;
  
• Forceps;
  
• pH meter;
  
• distilled water in wash bottle;
  
• 15 milliliter conical polypropylene capped and sterile tubes;
  
• 2 liter glass beaker;
  
• distilled water;
  
• stir bar;
  
• magnetic stir plate;
  
• refrigerator;
  
• freezer;
  
• lab area with electricity, plumbing and proper lighting (a lot of people take these for granted. Hey...
  
  I don't want to assume anything. For all I know...
  
  There might be some lone scientist operating 'out and away from civilization' that is starting from scratch in which case...
  
  I should add:
  
  
  • lab coat (optional),
    
  • shoes (optional), and
    
  • proper protective eye-wear (Absolutely critical! Why? While I wish to protect the scientist I'm also highly motivated to 'cover my derriere' since I don't live in a 'lone-away-from-civilization locale' but in a highly litigious U.S. of A. climate. Capeeeeesh?)
  
  
  
• 95% Ethanol;
  
• 1M Tris pH 6.0;
  
• 1M Tris pH 7.5;
  
• 1M Tris pH 8.0;
  
• 5M NaCl
  
• 1X TBS solution = 10 mM Tris pH 7.5 and 500mM NaCl = 500 microliters of 1 M Tris pH 7.5 and 5 milliliters of 5 M NaCl and 44.5 millilites of distilled water;
  
• plastic, 'kitchen grade' storage containers to soak your membranes;
  
• paper towels to clean up spills and dry your hands and labware (post clean-up)
  
• Carnation Dry Milk (or other generic brand of powdered milk);
  
• 1% Thimerosal solution (Sodium salt of 2-ethylmercuriomercaptobenzoic acid = sodium etylmercurithiosalicylate = Mercury-([o-carboxyphenyl]thio)ethyl sodium salt = C₉H₉HgO₂SNa having a formula weight of 404.8 grams per mole; 1% Solution = 1 g or thimerosal in 100 ml of distilled water; used as antibacterial and antifungal preservative);
  
• Tube rotating device;
  
• lab timer;
  
• Volumetric dispensing devices such as Pipetman pipettors;
  
• 10 mM Tris pH 7.5 (1 milliliter of 1M Tris pH 7.5 in 99 milliliters of distilled water);
  
• Glycine powder;
  
• Concentrated HCl;
  
• digital balance with taring capability;
  
• 100 mM Glycine pH 2.5 (Add 375 milligrams of glycine powder into a 50 milliliters conical tube and bring volume up to 45 milliliters; adjust pH to 2.5 by adding about 100 microliters of concentrated HCl, bring final volume up to 50 milliliters, cap tube and shake to mix);
  
• 100 mM triethylamine pH 11.5 (make fresh just before you need it and here's how you make it: place a clean 50 ml conical tube in a 150 ml glass beaker and set the beaker with nested tube on the pan of a balance; zero out the read-out by pressing on the tare button; add 0.5 g of liquid triethylamine using a volumetric dispensing device; add distilled water to the 50 ml mark; cap the conical tube and invert to mix);
  
• 10 mM Tris pH 6.0 (1 ml of 1M Tris pH 6.0 plus 99 ml of distilled water)
  
• Slide-A-Lyzer Dialysis Cassette (0.5 ml to 3.0 ml capacity and 3,500 KDa cut-off) from Pierce Scientific Product [www.piercenet.com] Item#: 66330);
  
• 1.5 ml conical, capped microfuge tubes;
  

If you can get the above materials ready, ahead of time, you'll be able to carry out the affinity purification procedure much smoother than if you have to create stock solutions and working solutions 'on-the-fly'.

In the next post I will give you the sequential steps (the instructions) which I have followed to successfully affinity purify rabbit antibodies against my 'protein of interest' (i.e. the protein used to immunize the test animals... which happened to be rabbits in my case).

From a computer in Central New York, this is LeBlond, Gerard LeBlond advising you to 'go for the gusto!'


P.S. Please, no comments or complaints from anyone named 'Gusto'. Thank you.

Technique To Affinity Purify Antibodies From Immunized Animal Sera Part 1

Hello,

My name is LeBlond, Gerard LeBlond and my purpose is to serve up detailed techniques that can be used to perform specific tasks in the Molecular Biology lab.

Recently I was commissioned with the task of raising antibodies against a new protein discovered in our lab.

Whose lab made this request? The research group is headed by David R. Mitchell, Ph.D. (principal investigator) of the Department of Cell & Developmental Biology at SUNY UMU in Syracuse, NY.

In this 4 part series of posts I shall reveal the protocol that I used to accomplish that task.

But first I should mention that the procedure, called: "Affinity Purification of Antibody From Sera" is meaningless by itself and in isolation from any other work.

For instance, in order to begin the project it is first necessary to isolate the previously uncharacterized protein (or you can start with a previously isolated protein and do the technique for practice... but what's the fun in that?), then you need to immunize lab animals (we used rabbits) using a specific protocol and you need to make certain you isolate blood serum prior to (this is, by convention, called the 'pre-bleed' sera though it could just as accurately be called the 'pre-immune' sera... but who am I to fight convention) exposing the animal to your protein (the antigen), and you need to take blood samples periodically after each antigenic boost.

You'll also need to size-separate your protein using SDS-polyacrylamide gel electrophoresis and adhere your protein onto an hydrophobic membrane using the Western Transfer procedure.

In subsequent posts I'll go over the policies and procedures associated with successfully performing each of those techniques but this series of missives is dedicated to affinity purification.

In Part 2 of the series of posts I will explain exactly what you need before you begin the procedure.

Part 3 will be dedicated to giving the sequential steps which I have followed to successfully affinity purify rabbit antibodies against my newly discovered protein.

And Part 4 will focus on providing helpful hints for making the process run efficiently and some of the tricky areas to pay particular attention.

So, from Central New York, this is LeBlond, Gerard LeBlond.


P.S. In order to respect the confidentiality and certain priveleged information between my associates I can not divulge the identity or specific characteristics of the newly discovered protein. That information can only see the light of day via the process of peer-reviewed publication in a mainstream scientific journal.

P.P.S. Of course, when the results are published I will be happy to quote the name of the publication and the dates and pages associated with the published work. But until then the word is "MUM".