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組織染色でうまくつかえる抗GAL4 抗体
Date: Fri, 24 Sep 1999 15:20:47 +0900
From: Tomokazu Ohshiro
Subject: [Jfly] 組織染色用抗GAL4 抗体
Jfly の皆様、
どなたか組織染色でうまくつかえる抗GAL4 抗体について御存じでしたら、
教えていただけませんでしょうか？用途はハエのGAL4ラインで、GAL4の
発現領域を見るためです。
東北大、加齢医学研究所、神経機能情報部
大城 朝一
X-Sender: tohshiroアットマークeuropa.idac.tohoku.ac.jp
Mime-Version: 1.0
Date: Wed, 29 Sep 1999 13:52:19 +0900
X-Original-To: jflyアットマークnibb.ac.jp
From: Tomokazu Ohshiro
Subject: [Jfly] 組織染色用抗GAL4 抗体について
Reply-To: jflyアットマークnibb.ac.jp
Sender: itokeiアットマークnibb.ac.jp
To: jflyアットマークnibb.ac.jp
Jfly の皆様、
遺伝研の広海さんからanti-GAL4 抗体についてコメントを頂きました。
有益な情報も含まれておりますので、転送いたします。
クローンテックのモノクロ(anti-GAL4 DNA binding domain) を試された方は多いよ
うですが、うまくいったり、いかなかったりする例が多いようです。
我々の研究室でも試したのですが、いまいち染まりがよくないので他を探そうと
いう結論になりました。
それから広海さんからのresponce で少し触れています、anti-GAL4 activation
domain を試された方はいらっしゃいませんか？
大城
以下、広海さんからのお返事
大城朝一様、
Jfyでの議論（http://jfly.nibb.ac.jp/html/discussion/9508_anti_GAL4_antibody）
では実際に使ったことのある人はいないようですね。以前、Claudio Pikielnyが
biodrosに同じ質問をのせた時、彼に送った解答を転送します。
広海
＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝
From: Yasushi Hiromi
Subject: Re: [bio.dros] anti-Gal4?
Dear Claudio,
I've used a GAL4 Ab from Clontech (GAL4 DNA-BD mAb, Cat# 5399-1). It is
made against the DNA binding domain of GAL4. It works on tissue, but I
haven't suceeded in Westerns. Clontech also sells an antibody against the
GAL4 activation domain, and there is at least one more company (Santa Cruz)
that sells anti GAL4. The one from Santa Cruz did not work for me, and I
haven't tested the anti-GAL4 AD from Clontech.
I've added my correspondences with Clontech about how it worked. I hope it
would be of some use to you.
Regards,
Yash Hiromi
_____________________________________
Date: 9/25/95
To: Adrianne Horowitz
Clontech Technical Service
From: Yasushi Hiromi
Number of pages: 1
Dear Adrianne,
Re: GAL4 DNA-BD mAb, Cat# 5399-1.
Thank you very much for sending a sample of your GAL4 DBD antibody. We have
used it to address the following questions:
(1) Does this MAb recognize fusion proteins that only contain the first 74
amino acids of GAL4?
(2) Does this antibody detect antigens in tissue using indirect
immunofluorescence or imunohistochemistry, or does it only work on Western
blots?
We have answers to both questions: the first one is NO, the second one is YES.
10/2/95
Dr. Paul Diehl
Clontech Technical Service
Dear Paul,
Enclosed are some photographs of Drosophila eye imaginal discs
stained with your antibody against.GAL4 DBD (cat# 5399-1). The antibody was
preabsorbed with wild type embryos and was used at 1:3000 dilution. The
secondary antibody was Goat anti-mouse IgG-HRP conjugated (Jackson). Tissue
was fixed with PLP for 30 minutes on ice. The promoter that drives the GAL4
fusion genes is expressed in all cells posterior to the morphogenetic furrow
(Hay et al., Development 120, 2121-9 [1994]). The morphogenetic furrow is
an indentation in the disc epithelium that moves in a posterior to anterior
direction; in these preparations, the furrow is approximately half way along
the anterior-posterior dimension (for a description of Drosophila eye
development, see Tomlinson, A. Development 104, 183-193 [1988]). The
animals used in this experiment carry GAL4 fusion transgenes that were
inserted into the genome by P-element mediated germline transformation. You
can see that those expressing
fusions containing GAL4 1-147 stain, but those that contain only GAL4 1-74
do not stain.
If you have any questions about these results, please feel free to
e-mail or call me. Thanks again for your sample.
Sincerely yours,
Yasushi Hiromi
Assistant professor of molecular biology