JPS63206658A - High sensitivity detection of protein - Google Patents
High sensitivity detection of proteinInfo
- Publication number
- JPS63206658A JPS63206658A JP3995387A JP3995387A JPS63206658A JP S63206658 A JPS63206658 A JP S63206658A JP 3995387 A JP3995387 A JP 3995387A JP 3995387 A JP3995387 A JP 3995387A JP S63206658 A JPS63206658 A JP S63206658A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- membrane
- palladium
- electroless plating
- colloid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 34
- 230000035945 sensitivity Effects 0.000 title claims abstract description 10
- 238000001514 detection method Methods 0.000 title claims description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000012528 membrane Substances 0.000 claims abstract description 23
- 229910052763 palladium Inorganic materials 0.000 claims abstract description 19
- 239000000084 colloidal system Substances 0.000 claims abstract description 15
- 239000010953 base metal Substances 0.000 claims abstract description 8
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 abstract description 26
- 238000007747 plating Methods 0.000 abstract description 11
- 238000007772 electroless plating Methods 0.000 abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 10
- 230000003197 catalytic effect Effects 0.000 abstract description 8
- 239000007788 liquid Substances 0.000 abstract description 6
- 238000000366 colloid method Methods 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 229910052751 metal Inorganic materials 0.000 abstract description 4
- 239000002184 metal Substances 0.000 abstract description 4
- 239000002245 particle Substances 0.000 abstract description 4
- 238000007654 immersion Methods 0.000 abstract description 2
- 230000003100 immobilizing effect Effects 0.000 abstract description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 5
- 229910052709 silver Inorganic materials 0.000 description 5
- 239000004332 silver Substances 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 230000001235 sensitizing effect Effects 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- -1 polyethylene Polymers 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000002940 palladium Chemical class 0.000 description 1
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000001476 sodium potassium tartrate Substances 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は新規なタンパク質の高感度検出法に関するもの
である。さらに詳しくいえば２本発明は。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel highly sensitive detection method for proteins. To be more specific, there are two aspects of the present invention.
膜上に固定されたタンパク質を簡単な手段で、安全かつ
経済的に、しかも金コロイド法に匹敵するような高感度
で検出しうる実用的な方法に関するものである。The present invention relates to a practical method for detecting proteins immobilized on membranes by a simple means, safely and economically, and with high sensitivity comparable to the gold colloid method.
従来の技術
従来、タンパク質や核酸の分析法として電気泳動法が知
られており、この電気泳動法の中でも。Conventional technology Electrophoresis has been known as a method for analyzing proteins and nucleic acids.
特にポリアクリルアミドゲル、アガロースゲルなどを利
用したゲル電気泳動法は、簡単な装置で多くの情報が得
られる上に、サンプル量も少なくてよいという利点を有
することから、生化学や医学の分野で重要な手法として
幅広く用いられている。In particular, gel electrophoresis using polyacrylamide gel, agarose gel, etc. has the advantage of obtaining a lot of information with a simple device and requiring only a small amount of sample, so it is widely used in the fields of biochemistry and medicine. It is widely used as an important method.
特に、近年、ゲル内の物質をニトロセルロースなどの膜
上に固定するプロッティングの技術が開発されて、免疫
的な手法や核酸のハイブリダイゼイションを利用する手
法を使用しうるようになったことにより、さらに一枚の
ゲルから複数の転写した膜が得られ、染色法を変えての
相互比較ができるようになったことにより、飛躍的に多
くの情報が得られるようになった。In particular, in recent years, plotting techniques have been developed in which substances in gels are immobilized on membranes such as nitrocellulose, making it possible to use immunological techniques and techniques that utilize nucleic acid hybridization. As a result, multiple transferred membranes can be obtained from a single sheet of gel, and mutual comparisons can be made using different staining methods, making it possible to obtain dramatically more information.
ところで、膜上のタンパク質などの検出法としては、従
来、アミドブラックやクマジーブルーなどの有機色素を
用いる方法が知られているが、前記のような技術の進展
に伴い、より高感度な検出法が要求されるようになり９
例えば金コロイドのタンパク質や核酸への吸着性を利用
して一次染色したのち、吸着された金コロイドを核に銀
を析出させて増感する方法が開発された。By the way, methods using organic dyes such as amide black and Coomassie blue are conventionally known as methods for detecting proteins on membranes, but with the development of the above-mentioned technology, more sensitive detection Laws are now required9
For example, a method has been developed in which primary staining takes advantage of colloidal gold's ability to adsorb proteins and nucleic acids, and then sensitization is achieved by depositing silver on the cores of the adsorbed colloidal gold.
しかしながら、このような金コロイド法においては、極
めて高価な金が用いられ、また銀増感用の試薬は光安定
性が低く、取り扱いに極めて高度な技術が要求されるな
どの欠点がある。However, such a gold colloid method has drawbacks such as extremely expensive gold is used, the reagent for silver sensitization has low photostability, and extremely sophisticated techniques are required for handling.
近年、電気泳動法の臨床検査への応用が盛んに研究され
ているが、この場合は、単なる研究用に比べて、より安
全、安価、簡単であることが要求されるので、ラジオア
イソトープの使用や水銀などの使用は好ましくなく、ま
た使用量の割には高価であり、かつ光安定性の低い銀増
感試薬も臨床用としては適当とはいえない。In recent years, the application of electrophoresis to clinical testing has been actively researched, but in this case, the use of radioisotopes is required because it is required to be safer, cheaper, and simpler than for mere research. The use of silver or mercury is not preferred, and silver sensitizing reagents that are expensive relative to the amount used and have low photostability are also not suitable for clinical use.
発明が解決しようとする問題点
本発明の目的は、このような事情のもとで、膜上に固定
されたタンパク質を簡単な手段で、安全かつ経済的に、
しかも金コロイド法に匹敵するような高感度で検出する
ための実用的な方法を提供することにある。Problems to be Solved by the Invention Under these circumstances, the purpose of the present invention is to safely and economically immobilize proteins on membranes by simple means.
Moreover, the object is to provide a practical method for detection with high sensitivity comparable to the gold colloid method.
問題点を解決するための手段
本発明者らは前記目的を達成するために鋭意研究を重ね
た結果、パラジウムコロイド粒子がタンパク質に吸着さ
れやすいこと及びパラジウムが触媒活性を有することに
着目し、タンパク質にパラジウムコロイドを吸着させ、
これに卑金属を無電解めっきして増感することによりそ
の目的を達成しつることを見出し、この知見に基づいて
本発明を完成するに至った。Means for Solving the Problems As a result of extensive research to achieve the above object, the present inventors focused on the fact that palladium colloidal particles are easily adsorbed to proteins and that palladium has catalytic activity. Adsorb palladium colloid to
It was discovered that the object could be achieved by sensitizing it by electroless plating with a base metal, and based on this knowledge, the present invention was completed.
すなわち２本発明は、膜上に固定されたタンパク質を検
出するに当り、破膜をノニオン性界面活性剤で安定化し
たパラジウムコロイド液で処理し。Namely, in the present invention, when detecting proteins immobilized on a membrane, a broken membrane is treated with a palladium colloid solution stabilized with a nonionic surfactant.
次いでこれに卑金属無電解めっきすることを特徴とする
タンパク質の高感度検出法を提供するものである。The object of the present invention is to provide a highly sensitive method for detecting proteins, which is characterized in that this is then electrolessly plated with a base metal.
本発明方法において、被検出タンパク質の固定に用いら
れる膜としては、ニトロセルロースメンブランなど、金
コロイド法などにおいて慣用されているものを使用する
ことができる。また、該膜上に被検出タンパク質を固定
する方法としては。In the method of the present invention, membranes commonly used in colloidal gold methods, such as nitrocellulose membranes, can be used as the membrane used to immobilize the protein to be detected. Also, as a method for immobilizing a protein to be detected on the membrane.
痛常行われている方法９例えばエレクトロプロッティン
グ法などが用いられる。A commonly used method 9 such as electroplotting method is used.
本発明方法において用いられるパラジウムコロイド液は
、ノニオン性界面活性剤で安定化される。The palladium colloidal liquid used in the method of the present invention is stabilized with a nonionic surfactant.
また、少量の吸着促進剤を加えることも有効である。ノ
ニオン性界面活性剤としては２例えばポリエチレンクリ
コール−ｐ−ノニルフェニルエーテルなど、吸着促進剤
としては、酢酸、ギ酸などが挙げられる。このパラジウ
ムコロイド液中のパラジウムの含有量は２通常０．０１
〜３０ｍｍｏｌ／ノの範囲で選ばれ、一方前記ノニオン
性界面活性剤の含有量は０．００１〜５重量％の範囲で
選ばれる。It is also effective to add a small amount of adsorption promoter. Examples of the nonionic surfactant include polyethylene glycol-p-nonylphenyl ether, and examples of the adsorption promoter include acetic acid and formic acid. The content of palladium in this palladium colloidal liquid is 2 usually 0.01
The content of the nonionic surfactant is selected within the range of 0.001 to 5% by weight.
吸着促進剤は、塩酸などの無機酸、ピリジン。Adsorption promoters include inorganic acids such as hydrochloric acid and pyridine.
エタノール、メタノールなどの有機溶媒、酢酸。Organic solvents such as ethanol, methanol, acetic acid.
ギ酸などの有機酸が有効でコロイド液量の０．０１〜１
ｔＸ）程度が好ましい。Organic acids such as formic acid are effective and the amount of colloid liquid is 0.01 to 1.
tX) is preferable.
該パラジウムコロイド液の調製は１例えばノニオン性界
面活性剤の存在下で、パラジウム錯体を水素化ホウ素ナ
トリウム水溶液で還元することにより行われる。The palladium colloidal liquid is prepared, for example, by reducing the palladium complex with an aqueous sodium borohydride solution in the presence of a nonionic surfactant.
＝４− パラジウムコロイド粒子が吸着して染色される。=4- Palladium colloid particles are adsorbed and dyed.
この処理のみでもタンパク質の検出は可能であるが、感
度が低くて、スポット当りＬｏｎｇ程度までしか検出す
ることができない。Although it is possible to detect proteins with this treatment alone, the sensitivity is low and only a long range can be detected per spot.
したがって１本発明においては、前記のようにして処理
した膜に、さらに卑金属を無電解めっきして増感する。Therefore, in one aspect of the present invention, the film treated as described above is further sensitized by electroless plating with a base metal.
一般に、無電解めっきは、置換めっき、接触めっき、非
触媒化学めっき及び触媒化学めっきに分けられるが９本
発明の無電解めっきは、パラジウムの触媒活性剤を利用
する触媒化学めっきであり。Generally, electroless plating is divided into displacement plating, contact plating, non-catalytic chemical plating, and catalytic chemical plating.9 The electroless plating of the present invention is catalytic chemical plating that utilizes a palladium catalyst activator.
これを用いると、触媒作用のある金属面のみに選択的に
金属を析出させることができる。このめっき液に含有さ
せる卑金属としては２通常ニッケルや銅が好ましく、こ
れらは水溶性塩の形で加えられる。When this is used, metal can be selectively deposited only on metal surfaces that have a catalytic effect. The base metal contained in this plating solution is usually preferably nickel or copper, which is added in the form of a water-soluble salt.
本発明においては、無電解めっき液中に浸せきする処理
を行うことにより、被検出タンパク質に該ハラタウムコ
ロイド粒子上に析出するので、該タンパク質を高感度で
検出することができる。この方法によるとスポット当り
０．１ｄｇ程度のタンパク質まで検出することが可能で
ある。In the present invention, the protein to be detected is precipitated on the Harataum colloid particles by immersion in an electroless plating solution, so that the protein can be detected with high sensitivity. According to this method, it is possible to detect up to about 0.1 dg of protein per spot.
発明の効果
本発明方法によると、膜上に固定化されたタンパク質を
簡単な手段で、安全かつ経済的に、しかも金コロイド法
に匹敵するような高感度で検出することができる上に、
無電解めっき液は、金コロイド法で用いられている銀増
感用試薬と異なり。Effects of the Invention According to the method of the present invention, proteins immobilized on membranes can be detected by simple means, safely and economically, and with high sensitivity comparable to the gold colloid method.
Electroless plating solution is different from the silver sensitizing reagent used in the colloidal gold method.
常温で長期間の保存に耐えうるなどの利点を有している
。It has the advantage of being able to withstand long-term storage at room temperature.
このような優れた特徴を有する本発明方法は極めて実用
的価値の高い方法といえる。The method of the present invention having such excellent features can be said to be of extremely high practical value.
実施例 次に実施例によって本発明をさらに詳細に説明する。Example Next, the present invention will be explained in more detail with reference to Examples.
ポリエチレングリコール−ｐ−ノニルフェニルエーテル
の存在下に、塩化パラジウム（川を水素化ホウ素ナトリ
ウム水溶液で還元して、パラジウム含有量が０．５ ｍ
ｍｏｌ ／ ｌｊのパラジウムコロイド液ヲ調製した。In the presence of polyethylene glycol-p-nonylphenyl ether, palladium chloride was reduced with an aqueous sodium borohydride solution to a palladium content of 0.5 m
A palladium colloid solution of mol/lj was prepared.
また９次の組成を有するめっき液を調製した。A plating solution having a 9th order composition was also prepared.
酒石酸ナトリウムカリウム ０．８
水酸化ナトリウム ０．８
ホルムアルデヒド ６．３
代表的タンパク質として牛血清アルブミンを濃度０．０
１〜１００兜に希釈した液１０μｌを、膜上にキャピラ
リでスポットして（約５ｍｍ径）試験片を作成した。Sodium potassium tartrate 0.8 Sodium hydroxide 0.8 Formaldehyde 6.3 Bovine serum albumin as a representative protein at a concentration of 0.0
A test piece was prepared by spotting 10 μl of the solution diluted to 1 to 100 μl onto the membrane using a capillary (about 5 mm in diameter).
この試験片を、前記のパラジウムコロイド１０コに１２
２時間浸セキることによりｙ ２０ ｎｇ／ ５ｐｏ
ｔの蛋白まで染色により視認できるようになった。This test piece was added to 10 pieces of the palladium colloid mentioned above for 12 hours.
y 20 ng/5po by soaking for 2 hours
It became possible to visually recognize the t protein by staining.
同じ＜１０１１Ｌｌのコロイドと、それに、吸着促進剤
として０．５ ｍ／のピリジン、ＩＮ塩酸、または酢酸
を加え、３０分間浸せきした場合、無添加コロイドでは
、全くタンパク質は検出されなかったが。When the same <1011 Ll of colloid was added with 0.5 m/ml of pyridine, IN hydrochloric acid, or acetic acid as an adsorption promoter and soaked for 30 minutes, no protein was detected with the colloid without any additives.
ピリジンまたはＩＮ塩酸を加えた場合は１１００ｎ／
５ｐｏｔ 、酢酸の場合は、 １０ ｎｇ／ ５ｐｏ
ｔ までのタンパク質を視認できた。If pyridine or IN hydrochloric acid is added, 1100n/
5pot, for acetic acid, 10 ng/5pot
Proteins up to t were visible.
同様に、ｌＱｄのコロイドに５ｄのメタノールを加え、
１時間浸せきした時はｅ ５０ ｎｇ／ ５ｐｏｔ、
２μｌのギ酸を加え１時間浸せきした時は５ ｎｇ／
５ｐｏｔ。Similarly, add 5d methanol to lQd colloid,
When soaked for 1 hour, e 50 ng/5pot,
When 2 μl of formic acid was added and immersed for 1 hour, 5 ng/
5 pots.
メタノール１ｍｌ、ギ酸５０μノを加え、３０分浸セキ
した時は１ ｎｇ／５ｐｏｔのタンパク質を視認できた
。When 1 ml of methanol and 50 μm of formic acid were added and soaked for 30 minutes, 1 ng/5 pot of protein was visible.
同様に、１０ｍのコロイドに１０μノの酢酸を加え、１
時間浸セキしたものは、２ｄｇ／５ｐＯｔまでのタンパ
ク質を視認できたが、これを水洗後すぐに。Similarly, add 10μ of acetic acid to 10m of colloid,
Proteins up to 2 dg/5 pOt were visible in the samples that had been soaked for a while, but this was immediately washed with water.
銅を含むめっき液に浸し、再度水洗、乾燥したところ＊
０．１ ｎｇ／５ｐｏｔまでの、すべてのスポット
でタンパク質を検出することができた。After soaking in a plating solution containing copper, washing again with water, and drying*
Protein could be detected in all spots up to 0.1 ng/5pot.
１０００ ｎｇ〜０．１ ｎｇまでの量の牛血清アルブ
ミンを常法に従い、ＳＤＳ電気泳動後、ニトロセルのポ
リエチレングリコール−ｐ−ノニルフェニルエーテルと
０．０５％の酢酸を含むコロイド中に４時間浸せき、再
度水洗し、銅を含むめっき液で増感したところ０．１
ｎｇまでのすべてのレーンで、牛血清アルブミンのバン
ドが視認された。これは。After SDS electrophoresis, bovine serum albumin in an amount of 1000 ng to 0.1 ng was immersed in Nitrocell's colloid containing polyethylene glycol-p-nonylphenyl ether and 0.05% acetic acid for 4 hours according to a conventional method. After washing with water again and sensitizing with a plating solution containing copper, the result was 0.1
A bovine serum albumin band was visible in all lanes up to ng. this is.
アルドブラックで染色した場合の５００倍以上の感度で
あり、タンパク質のロード量の多いレーンでは、アミド
ブラック法ではほとんど認められなかった不純物のバン
ドが４本以上視認された。The sensitivity was more than 500 times that of staining with Aldoblack, and in lanes with a large amount of protein loaded, four or more bands of impurities, which were hardly observed with the Amidoblack method, were visible.
Claims (1)
膜をノニオン性界面活性剤で安定化したパラジウムコロ
イド液で処理し、次いでこれに卑金属を無電解めっきす
ることを特徴とするタンパク質の高感度検出法。1. In detecting proteins fixed on a membrane, the membrane is treated with a palladium colloid solution stabilized with a nonionic surfactant, and then a base metal is electrolessly plated on the membrane. Sensitivity detection method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3995387A JPS63206658A (en) | 1987-02-23 | 1987-02-23 | High sensitivity detection of protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3995387A JPS63206658A (en) | 1987-02-23 | 1987-02-23 | High sensitivity detection of protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63206658A true JPS63206658A (en) | 1988-08-25 |
| JPH0563744B2 JPH0563744B2 (en) | 1993-09-13 |
Family
ID=12567322
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3995387A Granted JPS63206658A (en) | 1987-02-23 | 1987-02-23 | High sensitivity detection of protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63206658A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0421234A2 (en) * | 1989-09-27 | 1991-04-10 | Abbott Laboratories | Hydrophilic laminated porous membranes and methods of preparing same |
| EP0421235A2 (en) * | 1989-09-27 | 1991-04-10 | Abbott Laboratories | Hydrophilic laminated porous membranes and methods of preparing same |
| JPH03120468A (en) * | 1989-09-27 | 1991-05-22 | Abbott Lab | Porous diaphragm apparatus for chromato- graphic assay and method of manufacturing the same |
| JP2011505342A (en) * | 2007-11-21 | 2011-02-24 | バイオ−ラッド ラボラトリーズ，インコーポレイティド | Photoluminescent metal complexes for protein staining |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59221665A (en) * | 1983-05-16 | 1984-12-13 | イ−ストマン コダック カンパニ− | Quantitative determination method of macromolecule |
| JPS6118945A (en) * | 1984-07-05 | 1986-01-27 | Fuji Photo Film Co Ltd | Color diffusion transfer film unit |
-
1987
- 1987-02-23 JP JP3995387A patent/JPS63206658A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59221665A (en) * | 1983-05-16 | 1984-12-13 | イ−ストマン コダック カンパニ− | Quantitative determination method of macromolecule |
| JPS6118945A (en) * | 1984-07-05 | 1986-01-27 | Fuji Photo Film Co Ltd | Color diffusion transfer film unit |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0421234A2 (en) * | 1989-09-27 | 1991-04-10 | Abbott Laboratories | Hydrophilic laminated porous membranes and methods of preparing same |
| EP0421235A2 (en) * | 1989-09-27 | 1991-04-10 | Abbott Laboratories | Hydrophilic laminated porous membranes and methods of preparing same |
| JPH03120470A (en) * | 1989-09-27 | 1991-05-22 | Abbott Lab | Porous diaphragm apparatus for chromato- graphic assay and method of manufacturing the same |
| JPH03120468A (en) * | 1989-09-27 | 1991-05-22 | Abbott Lab | Porous diaphragm apparatus for chromato- graphic assay and method of manufacturing the same |
| JPH03120469A (en) * | 1989-09-27 | 1991-05-22 | Abbott Lab | Porous diaphragm apparatus for chromato- graphic assay and method of manufacturing the same |
| JP2011505342A (en) * | 2007-11-21 | 2011-02-24 | バイオ−ラッド ラボラトリーズ，インコーポレイティド | Photoluminescent metal complexes for protein staining |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0563744B2 (en) | 1993-09-13 |
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