Method of detecting peptides and / or proteins

Abstract

The invention relates to a method of detecting peptides and / or proteins, comprising the steps of: contacting the peptides and / or proteins with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and detecting the peptides and / or proteins. The invention further relates to the use of such a composition for detecting peptides and / or proteins, and the corresponding kits. Preferred specific fields of use include polyacrylamide gel electrophoresis (PAGE).

Description

METHOD FOR DETECTING PEPTIDES AND / OR PROTEINS TECHNICAL FIELD

[0001] The present invention relates to the field of molecular biology and biochemistry, in particular the detection of peptides and / or proteins - and in particular protein analysis by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). BACKGROUND OF THE INVENTION

[0002] The detection of peptides and proteins represents an important task for numerous questions in the field, and there is a fundamental need for sensitive, efficient, rapid, reversible and / or, if possible, non-toxic methods for their detection - for example by means of staining.

[0003] However, various detection methods described in the prior art do not fulfill one or more of the above-mentioned aspects or do not fulfill them satisfactorily.

[0004] A special area of ​​application for protein detection is polyacrylamide gel electrophoresis (PAGE), which is well known in the field, and in particular SDS-PAGE, which is a widely used method for separating proteins according to their size. After separation, the proteins are usually made visible by staining and thus detected.

[0005] Known methods for staining proteins (e.g. in SDS-PAGE) include Coomassie staining, silver staining and fluorescent staining (see e.g. https: / / www.thermofisher.com / de / de / home / life-science / protein-biology / protein-biology-learning-center / protein-biology-resource-library / pierce-protein-methods / protein-gel-stains.html in the context of SDS-PAGE).

[0006] However, common protein staining methods (especially in SDS-PAGE) such as Coomassie or silver staining have disadvantages in terms of sensitivity, speed and compatibility with downstream applications.

[0007] For example, Coomassie staining is very simple, but can only detect protein quantities as low as 0.1 micrograms. A conventional Coomassie staining protocol typically takes more than two hours and requires the use of toxic and environmentally harmful chemicals such as methanol and acetic acid. Furthermore, Coomassie staining is usually irreversible or can only be reversed with considerable effort. This severely limits the subsequent analysis of the stained proteins, as techniques such as Western blotting or MALDI-TOF mass spectrometry cannot be applied.

[0008] Although highly sensitive, silver staining is a complex and costly process. It carries the risk of chemical modification of proteins, which can compromise subsequent applications such as MALDI-TOF or Western blotting.

[0009] Fluorescence staining, on the other hand, offers relatively high sensitivity and a wide linear dynamic range. However, it requires specialized equipment and expensive dyes, which also limits its application.

[0010] Furthermore, a "stain-free" staining method patented by BioRad, although described as quick and uncomplicated, also has limitations. It relies on a specific chemical modification of proteins with a toxic substance, namely trichloroethanol. Furthermore, only tryptophans form a fluorophore and are stained. This specificity limits the applicability of the method to proteins containing tryptophan. Consequently, proteins without this amino acid backbone cannot be detected using this method.

[0011] Accordingly, it is an object of the invention to provide methods for detecting peptides and / or proteins that are as sensitive, efficient, rapid, reversible and / or non-toxic as possible and that overcome the disadvantages described above.

[0012] The present invention aims, for example, to provide an improved staining method for proteins in connection with PAGE in order to overcome the disadvantages described above. SUMMARY OF THE INVENTION

[0013] The invention relates, inter alia, to the subject matter defined in the claims.

[0014] The invention relates, on the one hand, to methods for detecting peptides and / or proteins, comprising the steps of: bringing the peptides and / or proteins into contact with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and detecting the peptides and / or proteins.

[0015] Furthermore, the invention relates to the use of a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) for the detection of peptides and / or proteins.

[0016] The invention further relates, inter alia, to corresponding kits for the separation and detection of peptides and / or proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-C show SDS PAGEs of different BSA concentrations after 30 min of staining (A), after 60 min of staining (B) and after 60 min of destaining (C).

[0018] Figure 2 shows SDS PAGEs of different BSA concentrations after 60 min of staining.

[0019] Figures 3A and 3B show SDS-PAGE stained with Midori Green Advance (A) and a subsequent Western blot (B).

[0020] Figures 4A-D show SDS-PAGEs of BSA in different amounts stained with the following additives: Additive A: 1% DAPI in water, Additive B: 1% acridine orange and 1% DAPI in water, Additive C: Midori Green Advance, Additive D: 1% acridine orange in water. DESCRIPTION OF THE INVENTION

[0021] The invention particularly relates to the subject matter defined in the claims.

[0022] The present invention solves the above problem by the methods described in the claims and related objects, such as associated uses and kits.

[0023] In a first main aspect, the invention relates to methods for detecting peptides and / or proteins, comprising the steps of: contacting the peptides and / or proteins with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and detecting the peptides and / or proteins.

[0024] The invention also relates in particular to methods for detecting peptides and / or proteins, comprising the steps of: bringing the peptides and / or proteins into contact with Midori Green; and detecting the peptides and / or proteins.

[0025] In a second main aspect, the invention relates to the use of a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) for the detection of peptides and / or proteins.

[0026] The invention also relates in particular to the use of Midori Green for the detection of peptides and / or proteins.

[0027] The invention further relates, inter alia, to corresponding kits for the separation and detection of peptides and / or proteins.

[0028] In a third main aspect, the invention accordingly relates to a kit comprising a PAGE precast gel for the separation of peptides and / or Proteins; a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and optionally, instructions for use of the kit. The instructions for use preferably include instructions for contacting the composition with peptides and / or proteins.

[0029] The invention also relates in particular to such a kit, the composition of which is or contains Midori Green.

[0030] In an additional aspect, the invention relates to the use of the described kits for the detection of peptides and / or proteins.

[0031] In yet another aspect, the invention relates to a precast PAGE gel for the separation of peptides and / or proteins, comprising a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange).

[0032] In certain embodiments of the (aspects of) the invention, peptides are detected.

[0033] In the context of the invention, the peptides mentioned are not subject to any particular restrictions. Peptides are well known to those skilled in the art. They can, for example, be selected from the group consisting of oligopeptides (preferably comprising up to ten amino acid residues), polypeptides (preferably comprising 10 to 99 amino acid residues), and cyclopeptides (preferably comprising more than two amino acid residues).

[0034] In certain preferred embodiments of the invention, proteins are detected.

[0035] In the context of the invention, the proteins mentioned are not subject to any particular restrictions. Proteins are well known to those skilled in the art. They preferably comprise at least 100 amino acids. In certain (non-limiting) embodiments, the proteins have a size in the range of 10 kDa to 1000 kDa.

[0036] Acridine orange (N,N,N',N'-tetramethylacridine-3,6-diamine) is generally known to the person skilled in the art.

[0037] In certain preferred embodiments, a sample containing the peptides and / or proteins is contacted with said composition.

[0038] Methods and uses according to the invention may, in particular, further comprise the step of decolorizing the peptides and / or proteins. The decolorizing step preferably occurs after the detection step.

[0039] In certain embodiments of the invention, the peptides or proteins are separated by polyacrylamide gel electrophoresis (PAGE), such as SDS-PAGE.

[0040] Accordingly, the methods and uses according to the invention in certain preferred embodiments comprise the step of separating the peptides and / or proteins by means of polyacrylamide gel electrophoresis (PAGE)

[0041] Polyacrylamide gel electrophoresis (PAGE) is known to those skilled in the art and is not subject to any restrictions in the context of the present invention.

[0042] Polyacrylamide gel electrophoresis (PAGE) is particularly selected from the group consisting of native PAGE, SDS-PAGE, BAC-PAGE, CTAB-PAGE, urea-PAGE, and 2D gel electrophoresis. Preferably, the PAGE is SDS-PAGE.

[0043] If methods or uses according to the invention comprise a step of separating the peptides and / or proteins, the step of bringing into contact with the composition can in principle take place either before or after the separation step.

[0044] In certain embodiments, a sample containing the peptides and / or proteins is contacted with the composition prior to the separation step.

[0045] In certain embodiments, the peptides and / or proteins are contacted with the composition after the separation step, for example by contacting a corresponding PAGE gel with said composition.

[0046] Furthermore, the peptides and / or proteins may also be brought into contact with the composition during the separation step - for example, using a PAGE gel containing the composition.

[0047] Methods and uses according to the invention can also be used in particular in connection with Western blotting.

[0048] Thus, in certain embodiments, they further comprise the step of transferring the separated peptides and / or proteins from a PAGE gel to a membrane, wherein the transferring step occurs after the detection step, in particular wherein the method further comprises performing Western blotting.

[0049] According to the invention, the latter methods may also optionally comprise destaining the peptides and / or proteins. However, in certain more preferred embodiments, the latter methods do not comprise a step of destaining the peptides and / or proteins.

[0050] Methods and uses according to the invention can also be used in particular in connection with gel extraction.

[0051] Thus, in certain embodiments, they further comprise the step of removing at least one region from the PAGE gel, in particular wherein the method further comprises performing a gel extraction of the peptides and / or proteins present in the region.

[0052] In the course of their efforts, the present inventors have surprisingly first found that the The well-known DNA / RNA dye “Midori Green” is also very well suited for the detection of peptides or proteins - especially in light of the above task.

[0053] In addition, the present inventors have surprisingly found that a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) and 4',6-diamidine-2-phenylindole (DAPI) is very well suited for the detection of peptides or proteins.

[0054] In the course of their efforts, the present inventors have also surprisingly found in particular that N,N,N',N'-tetramethylacridine (contained in Midori Green) 3.6-diamine (acridine orange), is very well suited for the detection of peptides or proteins - and especially in light of the above task.

[0055] Without wishing to be bound by theory, it is assumed in the light of the present invention that by using acridine orange (N,N,N',N'-tetramethylacridine- 3.6-diamine) enables very efficient staining of peptides and proteins - whereby particularly good results can also be achieved in combination with other components of Midori Green.

[0056] In light of the above findings, the invention thus also relates in further embodiments (comprising a method) to the use of a composition comprising N,N,N',N'-tetramethylacridine- 3,6-diamine (acridine orange) and 4',6-diamidine-2-phenylindole (DAPI) for the detection of peptides and / or proteins.

[0057] Thus, the invention also provides, in particular, methods, uses and kits (as defined herein) in which said composition comprises N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) and 4',6-diamidine-2-phenylindole (DAPI).

[0058] The invention generally relates in further aspects, in particular also (comprising methods) to the use of Midori Green for the detection of peptides and / or proteins.

[0059] Thus, the invention also provides methods, uses, and kits (as defined herein) in which said composition is Midori Green. Likewise, the invention also specifically provides methods, uses, and kits (as defined herein) in which said composition contains Midori Green.

[0060] Midori Green is a commercially available dye well known to those skilled in the art, originally used to stain DNA or RNA. According to a study by Biotium, Inc., Fremont, CA, entitled "Comparison of Nucleic Acid Gel Stains; Cell permeability, safety, and sensitivity of ethidium bromide alternatives” by M. Guzaev et al., including acridine orange and DAPI (see, for example, https: / / biotiurn.com / wp-content / uploads / 2017 / 02 / Gel-Stains-Comparison.pdf).

[0061] Acridine orange (N,N,N',N'-tetramethylacridine-3,6-diamine) is well known to those skilled in the art as a nucleic acid binding dye that can stain both DNA and RNA in cells (see https: / / www.sciencedirect.com / topics / agricultural-and-biological-sciences / acridine-orange).

[0062] DAPI (4',6-diamidine-2-phenylindole) is a fluorescent dye also well known to those skilled in the art and has been described, among other things, to bind to the minor groove of DNA (cf. https: / / www.sciencedirect.com / topics / neuroscience / dap).

[0063] Midori Green is commercially available, for example, from the companies “NIPPON Genetics Europe GmbH” and “Biozym” as well as a number of other companies in Europe and worldwide (see e.g. www.nippongenetics.eu / distributors ).

[0064] Midori Green is offered in various variants that can be used in connection with the invention.

[0065] In certain embodiments of the various aspects of the invention, the said composition is thus preferably selected from the group consisting of Midori Green Advance, Midori Green Direct, Midori Green Xtra, Midori Green Easy, in particular wherein the composition is selected from the group consisting of Midori Green Advance and Midori Green Direct.

[0066] In preferred embodiments of the invention, Midori Green is used for detection.

[0067] In certain preferred embodiments herein, Midori Green Advance is used for detection. In certain preferred embodiments herein, Midori Green Direct is used for detection.

[0068] In the context of the invention, the detection of peptides or proteins (or the aforementioned detection step) is not subject to any particular restrictions. It can be readily carried out advantageously by a person skilled in the art.

[0069] In certain embodiments, detection occurs under green light excitation. In certain embodiments, detection occurs under blue light excitation.

[0070] In special embodiments, detection is carried out under excitation with light in the wavelength range of 480 - 530 nm.

[0071] In certain embodiments, detection occurs under UV light excitation.

[0072] In special embodiments, detection is carried out under excitation with light in the wavelength range of 280 - 315 nm, in particular in the range of approximately 300 - 305 nm, such as with light of a wavelength of approximately 302 nm.

[0073] Without wishing to be bound by theory, secondary peaks are observed at approximately 300 nm and 400 nm.

[0074] In further embodiments, detection is carried out under excitation with light in the wavelength range of 280 - 430 nm, in particular 300 - 400 nm, such as in the range of approximately 300 nm and / or approximately 400 nm.

[0075] Irrespective of these embodiments, the person skilled in the art can, in particular, also orient himself in the detection on the light types / wavelength ranges described in available protocols in connection with Midori Green by the commercial providers (see, for example, above) and, if necessary, easily optimize these for the specific purposes.

[0076] In still further aspects of the invention, the invention relates to the methods and uses described herein, wherein the term "detection" is replaced by the term "staining".

[0077] A number of certain preferred embodiments of the invention are listed in detail below:

[0078] That is, the invention also relates in particular to the following embodiments [1] to

[0036] :

[0079] [1]: A method for detecting peptides and / or proteins, comprising the steps of: contacting the peptides and / or proteins with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and detecting the peptides and / or proteins.

[0080] [2]: Method for the detection of peptides, comprising Steps: contacting the peptides with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and detecting the peptides.

[0081] [3]: Method for detecting proteins, comprising Steps: contacting the proteins with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and detecting the proteins.

[0082] [4]: Method according to one of the embodiments [1] to [3], characterized in that the detection takes place under green light excitation.

[0083] [5]: Method according to one of the embodiments [1] to [3], characterized in that the detection takes place under blue light excitation.

[0084] [6]: Method according to one of the embodiments [1] to [3], characterized in that the detection takes place under UV light excitation.

[0085] [7]: Method according to one of the embodiments [1] to [5], characterized in that the detection is carried out under excitation with light in the wavelength range of 480 - 530 nm.

[0086] [8]: Method according to one of the embodiments [1] to [3] and [6], characterized in that the detection is carried out under excitation with light in the wavelength range of 280 - 430 nm, in particular 300 - 400 nm.

[0087] [9]: Method according to one of the embodiments [1] to [8], further comprising the step of separating the peptides and / or proteins by polyacrylamide gel electrophoresis (PAGE).

[0088]

[0010] : Method according to embodiment [9], characterized in that the polyacrylamide gel electrophoresis (PAGE) is selected from the group consisting of native PAGE, SDS-PAGE, BAC-PAGE, CTAB-PAGE, urea PAGE and 2D gel electrophoresis.

[0089]

[0011] : Method according to embodiment

[0010] , characterized in that the polyacrylamide gel electrophoresis is 2D gel electrophoresis.

[0090]

[0012] : Method according to embodiment

[0010] , characterized in that the polyacrylamide gel electrophoresis is SDS-PAGE.

[0091]

[0013] : Method according to one of the preceding embodiments, characterized in that a sample containing the peptides and / or proteins is brought into contact with the composition.

[0092]

[0014] : Method according to one of the embodiments [9] to

[0013] , characterized in that the peptides and / or proteins according to the step of separation are brought into contact with the composition.

[0093]

[0015] : Method according to one of the embodiments [9] to

[0014] , further comprising the step of transferring the separated peptides and / or proteins from the PAGE gel to a membrane, in particular wherein the step of transferring takes place after the step of detection.

[0094]

[0016] : Method according to one of the embodiments [9] to

[0015] , characterized in that the method further comprises carrying out Western blotting.

[0095]

[0017] : Method according to one of the embodiments [9] to

[0014] , characterized in that the method further comprises the step of removing at least one region from the PAGE gel.

[0096]

[0018] : Method according to embodiment

[0017] , wherein the method further comprises performing a gel extraction of the peptides and / or proteins present in the region.

[0097]

[0019] : Method according to any one of the preceding embodiments, wherein said composition further comprises 4',6-diamidine-2-phenylindole (DAPI).

[0098]

[0020] : Method according to one of the preceding Embodiments, wherein said composition comprises Midori Green, in particular wherein the composition is selected from the group consisting of Midori Green Advance, Midori Green Direct, Midori Green Xtra, Midori Green Easy, further in particular wherein the composition is selected from the group consisting of Midori Green Advance and Midori Green Direct.

[0099]

[0021] : Method according to one of the preceding embodiments, wherein the method further comprises the step of destaining the peptides and / or proteins, wherein the destaining step preferably occurs after the detection step.

[0100]

[0022] Use of a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) for the detection of peptides and / or proteins.

[0101]

[0023] Use of a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) for the detection of peptides.

[0102]

[0024] Use of a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange) for the detection of proteins.

[0103]

[0025] : Use according to any one of embodiments

[0022] to

[0024] in a polyacrylamide gel electrophoresis method comprising the separation of the peptides and / or proteins.

[0104]

[0026] : Use according to one of the embodiments

[0022] to

[0025] , characterized in that said use is further characterized as defined in one of the embodiments [1] to

[0021] .

[0105]

[0027] : Use according to any one of embodiments

[0022] to

[0025] , wherein the composition is further characterized as defined in any one of embodiments

[0019] to

[0020] .

[0106]

[0028] A kit comprising a precast PAGE gel for the separation of peptides and / or proteins; and a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange).

[0107]

[0029] A kit comprising a precast PAGE gel for the separation of peptides; and a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange).

[0108]

[0030] A kit comprising a precast PAGE gel for the separation of proteins; and a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange).

[0109]

[0031] : Kit according to any one of embodiments

[0028] to

[0030] , wherein said composition is further characterized as defined in any one of embodiments

[0019] to

[0020] .

[0110]

[0032] : Kit according to any one of embodiments

[0028] to

[0031] , wherein the kit further comprises instructions for use of the kit, in particular wherein the instructions for use contain instructions for contacting the composition with peptides and / or proteins.

[0111]

[0033] A precast PAGE gel for the separation of peptides and / or proteins, comprising a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange), in particular wherein said composition is further characterized as defined in any one of embodiments

[0019] to

[0020] .

[0112]

[0034] : A method for detecting peptides and / or proteins, comprising the steps of: contacting the peptides and / or proteins with a composition containing Midori Green; and detecting the peptides and / or proteins, in particular further characterized as in any of the above embodiments [2] to

[0021] ,

[0113]

[0035] : Use of a composition containing Midori Green for the detection of peptides and / or proteins, in particular wherein the use is further characterized as described in any of the above embodiments [1] to

[0027] .

[0114]

[0036] : Kit comprising a precast PAGE gel for the separation of peptides and / or proteins; and a composition containing Midori Green; preferably further comprising instructions for use, in particular wherein the instructions for use contain instructions for contacting the composition with peptides and / or proteins; further preferably wherein the kit is further characterized as described in the above embodiments

[0029] to

[0032] . EXAMPLES OF IMPLEMENTATION

[0115] The following embodiments further explain the invention without, however, limiting it in any way.

[0116] Based on these examples, the present inventors have also discovered, among other things, that staining methods according to the invention are characterized, for example, by their speed (e.g., less than 1 hour), cost-effectiveness, and higher sensitivity compared to Coomassie staining (down to less than 50 ng BSA), with an even further increase in sensitivity appearing possible. According to the present inventors' results, the method is also fully reversible without significant additional effort, thus ensuring its compatibility with subsequent applications such as mass spectrometry or Western blotting.

[0117] Example 1 : Staining process and reversibility

[0118] Different amounts of BSA (bovine serum albumin) were dissolved in 20 μl of 1X Bolt™ LDS sample buffer and applied to a 12% Bolt™ Bis-Tris gel (Thermo Fisher Scientific). PAGE was run at 200 V for 22 minutes in 1X Bolt™ MOPS SDS running buffer. The gel was then incubated in 50 ml of TAE buffer containing 1 μl / ml Midori Green Direct. Incubation took place for a total of 60 minutes at room temperature with periodic rocking. (B) After 60 minutes of destaining in TAE buffer without Midori Green Advance, the gel was completely destained. (C) Figure 1 shows the resulting stained SDS-PAGE gel.

[0119] The complete reversibility of the staining enables subsequent methods such as Western blotting and mass spectrometry. This eliminates the need for complex destaining protocols and the use of toxic solvents often required for other staining methods, making the entire process safer and more efficient.

[0120] Example 2: Sensitivity

[0121] Different amounts of BSA (bovine serum albumin) were loaded onto an SDS-PAGE as described in Section 2.1 and stained with Midori Green. Figure 2 of the SDS-PAGE shows that protein amounts down to at least 50 ng can be successfully detected with Midori Green.

[0122] Example 3: Compatibility with Western Blotting

[0123] A fusion protein containing FGF2 was applied to SDS-PAGE as described in section 2.1 and stained with Midori Green. Detection was then carried out by Western blotting. For this purpose, the protein was transferred immediately after staining to a Thermo Scientific 0.45 pM nitrocellulose membrane for 60 minutes at a voltage of 100 V. The primary antibody used was anti-FGF2 antibody (Invitrogen, item number MA1-24682), which was diluted 1:1000 in I-Blot Blocking Solution. The membrane was incubated with the primary antibody for 60 minutes at RT while shaking. Detection was carried out using an AP-conjugated secondary antibody (Rockland Inc., item number 610-4541), which was diluted 1:2000 in I-Blot. The membrane was incubated in the secondary antibody solution for 60 minutes at RT. After washing three times in TBS-T, the membrane was developed with AP Staining Solution for 5 minutes. The resulting Western blot is shown in Figure 3.

[0124] The comparison in Figure 3 demonstrates that after staining an SDS-PAGE with Midori Green Advance, Western blot can be successfully performed without prior destaining. This represents a significant advantage over conventional methods such as Coomassie staining, which require destaining.

[0125] Example 4: Determination of the “active ingredients” of Midori Green

[0126] Different amounts of BSA (Bovine Serum Albumin) were dissolved in 20 μl of 1X Bolt™ LDS sample buffer and loaded onto a 12% Bolt™ Bis-Tris gel (Thermo Fisher Scientific). PAGE was run at 200 V for 22 minutes in 1X Bolt™ MOPS SDS running buffer. The gel was then incubated in 50 ml of TAE buffer containing 50 μl of various additives.

[0127] The incubation took place for a total of 30 minutes at Room temperature with periodic stirring and the following additives: Additive A: 1% DAPI in water, Additive B: 1% Acridine Orange and 1% DAPI in Water, Additive C: Midori Green Advance, Additive D: 1% acridine orange in water. Figure 5 shows the resulting stained SDS-PAGE gels. The proteins were illuminated with a FastGene Blue / Green LED flashlight.

[0128] Based on the comparison of the resulting SDS gels, it is assumed that acridine orange is already sufficient for the observed very efficient protein staining.

Claims

PATENT CLAIMS 1. A method for detecting peptides and / or proteins, comprising the steps: Contacting the peptides and / or proteins with a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange); and Detection of peptides and / or proteins.

2. Method according to claim 1, characterized in that the detection of the peptides and / or proteins takes place under green light, blue light or UV light excitation.

3. The method according to claim 1 or 2, further comprising the step of separating the peptides and / or proteins by polyacrylamide gel electrophoresis (PAGE).

4. The method according to claim 3, characterized in that the polyacrylamide gel electrophoresis (PAGE) is selected from the group consisting of native PAGE, SDS-PAGE, BAC-PAGE, CTAB-PAGE, urea PAGE and 2D gel electrophoresis, in particular wherein the polyacrylamide gel electrophoresis is SDS-PAGE.

5. Method according to one of the preceding claims, characterized in that a sample containing the peptides and / or proteins is brought into contact with the composition.

6. Method according to one of claims 3 to 5, characterized in that the peptides and / or proteins are brought into contact with the composition after the separation step.

7. The method according to any one of claims 3 to 6, further comprising the step of transferring the separated peptides and / or Proteins from the PAGE gel to a membrane, wherein the step of transferring occurs after the step of detection, in particular wherein the method further comprises carrying out Western blotting includes.

8. The method according to any one of claims 3 to 6, further comprising the step of removing at least one region from the PAGE gel, in particular wherein the method further comprises performing a gel extraction of the peptides and / or proteins present in the region.

9. A method according to any one of the preceding claims, wherein said composition i) further comprises 4',6-diamidine-2-phenylindole (DAPI); and / or ii) comprises Midori Green, in particular wherein the composition is selected from the group consisting of Midori Green Advance, Midori Green Direct, Midori Green Xtra, Midori Green Easy, further in particular wherein the composition is selected from the group consisting of Midori Green Advance and Midori Green Direct.

10. The method according to any one of the preceding claims, wherein the method further comprises the step of destaining the peptides and / or proteins, wherein the destaining step preferably occurs after the detection step. 1 1. Use of a composition containing N,N,N',N'- Tetramethylacridine-3,6-diamine (acridine orange) for the detection of peptides and / or proteins.

12. Use according to claim 11 in a polyacrylamide gel electrophoresis method comprising the separation of peptides and / or proteins.

13. Use according to any one of claims 11 to 12, wherein said composition is further characterized as defined in claim 9.

14. A kit comprising a precast PAGE gel for the separation of peptides and / or proteins; a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange), in particular wherein said composition is further characterized as defined in claim 9; and optionally, instructions for use of the kit.

15. A precast PAGE gel for the separation of peptides and / or proteins, comprising a composition comprising N,N,N',N'-tetramethylacridine-3,6-diamine (acridine orange), in particular wherein said composition is further characterized as defined in claim 9.

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