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Western blot analytical technique

A technique for blotting and analyzing results, applied in the field of western blotting analysis technology, which can solve problems such as errors, expensive photographic film processing components, and difficult maintenance

Active Publication Date: 2013-02-06
AGILENT TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Typically, photographic film processing components required to develop exposed film are expensive and difficult to maintain
Another problem with using photographic film is that obtaining unsaturated images of target proteins suitable for quantitative analysis requires time-consuming trial and error and is subject to error
[0012] Overall, standard western blotting methods are labor intensive, time consuming and use a large amount of reagents

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0085] gel electrophoresis

[0086] Prepare protein-containing samples as follows:

[0087] a. Incubate 20 μl protein sample with 20 μl fluorescent stain for 7 minutes at 75°C; and

[0088] b. Add 40 [mu]l loading buffer, mix and incubate at 75[deg.]C for an additional 5 minutes.

[0089] Additionally, control samples were prepared as follows:

[0090] a. Mix 20 μl protein sample with 10 μl reducing agent and 4x25 μl Invitrogen's LDS buffer; and

[0091] b. Incubate at 75°C for 5 minutes.

[0092] Both samples were loaded onto a NuPAGE electrophoresis gel and run according to the manufacturer's standard protocol to separate the proteins, and an image of the gel was then captured using a UV-transmitter and digital camera.

[0093] Transfer the separated sample to the membrane

[0094] The gel consisted of separated proteins divided into two equal parts and then one half was transferred to a PVDF membrane and the other half to a nitrocellulose membrane. Separated prote...

example 2

[0108] gel electrophoresis

[0109] Prepare protein-containing samples as follows:

[0110] a. Incubate 2 μl protein sample with 2 μl fluorescent stain for 7 minutes at 75°C;

[0111] b. Add 4 μl of loading buffer, mix and incubate at 75°C for 5 minutes; and

[0112] c. Add 2 μl of in-channel marker.

[0113] Load samples into Lab901P200 Electrophoresis gels were run according to the manufacturer's standard protocol to separate proteins. Use Lab901 for the used imaging (see Figure 5 "P200" in a).

[0114] Transfer the separated sample to the membrane

[0115] including isolated proteins From To recover, remove its carrier layer and use two blades to cut , exposing the top and bottom of the gel column contained within the 16 subcontainers. A comb comprising 16 gel pushing elements was used to push the gel within each sub-container so that the gel was drawn onto a PVDF membrane wetted in 20% methanolic Tris-Glycine transfer buffer. The membrane was placed ...

example 3

[0128] gel electrophoresis

[0129] Prepare protein-containing samples as follows:

[0130] a. Incubate 2 μl protein sample with 2 μl fluorescent stain for 7 minutes at 75°C;

[0131] b. Add 4 μl of loading buffer, mix and incubate at 75°C for 5 minutes; and

[0132] c. Add 2 μl of in-channel marker. Load samples into Lab901P200 Electrophoresis gels were run according to the manufacturer's standard protocol to separate proteins. Use Lab901 (see Figure 6 "P200" in a) used for imaging

[0133] Transfer the separated sample to the membrane

[0134] including isolated proteins From To recover, remove its carrier layer and use two blades to cut , exposing the top and bottom of the gel column contained within the 16 subcontainers. A comb comprising 16 gel pushing elements was used to push the gel within each sub-container so that the gel was drawn onto a PVDF membrane wetted in 20% methanolic Tris-Glycine transfer buffer. Membranes with a single protein transf...

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Abstract

The invention relates to a western blot analytical technique. In accordance with an embodiment of the present invention, there is provided a method of performing the western blot analytical technique, wherein the method comprises carrying out the following steps in the following order: a), pre-staining the proteins within a sample; b). separating the proteins using gel electrophoresis; c). analysing the separated proteins to determine the total protein load and / or at least one housekeeping protein load; d). transferring the proteins onto at least one membrane; e). probing the separated proteins to detect a target protein; and f). analysing the probed proteins to determine the target protein's molecular weight and / or load.

Description

[0001] This application claims priority on the filing dates of GB1008517.3, filed May 21, 2010, and GB1100092.4, filed January 5, 2011, the disclosures of which are incorporated herein by reference. technical field [0002] This patent application relates to improved methods for performing the technique of western blot analysis. Background technique [0003] Western blotting is a labor-intensive laboratory analytical method that is widely used in the life sciences to determine the presence and relative amount of a target protein in a complex sample. The term "target protein" is used to refer to a protein that the user of the analytical method desires to identify within a complex sample. Changes in protein expression (ie. upregulation and downregulation) are measured using relative amounts of proteins. [0004] Determining the presence or absence of a particular protein is achieved by combining two variables: the molecular weight of the protein and its immunological properti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N33/53G01N33/68G01N1/04
CPCG01N33/6803G01N27/44739G01N27/44726
Inventor 肯尼思·G·马克拿玛瑞斯图尔特·保尔沃特
Owner AGILENT TECH INC
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