Protein purification - experimental design | MedchemEXPRESS

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Protein purification - experimental design | MedchemEXPRESS

2021-11-30 06:05:51 39 ℃

Little M is not afraid to purify "difficult", IP, WB is just right. Bublied two-year laboratory small M, theory and practical experience, and see how I have "off" in the purification of protein.

First Off Product Selection

Small M knocks blackboard: The most important foundation of this level is also the most important, beware of "one step, step by step".

Affinity chromatography as a classic, efficient purification method, using the purpose of specific and reversible interaction between the molecule and the filler glycene, can be purified from the cell lysate or other biological sample, and a higher purity purpose molecule is obtained. Choose a suitable affinity purified agarose, which can be efficiently, convenient, reliably from biological samples, antibodies. How to choose "right"? And listen to me slowly:

■ Antibody basic knowledge articles, "knowing yourself know each other!"

Antibody, also known immunoglobulin (IMMunoglobulin, Ig) is a large Y-shaped glycoprotein produced by B cells. The antibody exists in one or more Y-shaped monomers, each Y-shaped monomer consists of four polypeptide chains: Heavy chain and two identical light chains (Figure 1) .

The specific sequence of the antibody heavy chain Fc region determines the type of Ig, such as α-IgA, δ-IgD, ε-IgE, γ-IgG, μ-IgM. There are two types of light chains, which are lambda-type and κ.

■ Antibody - ligand should match!

I am always the heart of human IgG3. After one pass, the destination zip position is empty. Finally, I found that PROTEIN A was chosen. Human IgG3 and Protein A, do not match! λ light chain and proten l, do not match! Protein purification also pays attention to door-to-door.

The affinity purified ligand of the antibody includes Protein A, Protein G, Protein L, and the like. For most mammals, Protein G has higher affinity than Protein A, such as human IgG3, mouse IgG1 and rat IgG2a, but Protein G can't be combined with human IgM, IGD, IGA.

Compared with Protein A, Protein G, Protein L has a more broad spectrum of IG binding capability, which can bind to all Ig (IgG, IgM, IgA, IgE and IGD) containing KAPPA light chain (IGD) and single-strandable variable fragment (SCFV) And FAB fragments. I would like to record the appropriate affinity purified ligand according to the type of antibody (Fig. 2).

Second custom method choice

MCE Protein A, Protein G, Protein L agarose, can separate antibodies from serum, cell culture supernatant or ascites; Anti-flag affinity gel can capture the target protein by IP or other methods; streptavidin Candy can be highly efficiently combined with biotinylated samples.

Antibody purification "big" experiment, IP, PULL DOWN "small" operation, gravity column "big" and from the heart tube "small". Kill you can use the slaughter knife, the product detailed use method (as follows only for reference only, see the instructions for each product).

■ Gravity cross-column method, suitable for separation, purifying antibodies from biological samples such as serum. Get a lot of antibodies in an experiment, wonderful!

1) Filling: Apply an appropriate amount of agarose filler according to the sample amount to add the affinity chromatography column, allowing the storage liquid to flow out; 2) Balance: BUFFER is balanced with the balance buffer; 3), to wash: Wash: After the balanced column is on, continue to be cleaned and removed by balance buffer. 4) Elution: Finally, elution of the destination sample was finally eluted into buffer.

■ Centrifugation method is suitable for interaction between proteins or study protein-proteins through IP capture proteins. Fine operation, stable!

Insert the Anti-flag affinity gel into the centrifuge tube, and use the centrifugal method. The centrifugation method is also washed out of the four steps, and the top three step buffer replacement is achieved by centrifugation, and finally collect the supernatant.

Third-level destination protein combination, elution

Input is clearly aimed, the protein is clearly combined, but there is no purpose strip in eluate. Skating a control, a suit, a protein eluting, I am in a line.

■ The smart woman is difficult to be no rice, I want me to "have no born"? NO WAY! There is no strip of the test results, perhaps the following reasons.

PS: Setting an Input control, determine the protein expression; retains the drainage / centrifugal supernatant, determine the sample has been combined. Each step is to "leave one hand", a comprehensive replication (analysis of experimental failure) is helpful.

■ "Vigor"? "Non-denatured"? Downstream experiment application to determine 1) Determination elution method This method eluting protein samples can be done directly to SDS-PAGE detection, simple and efficient. 2) Non-denatured elution method This method eluted protein samples maintain the original biological activity and can be used in post-functional analysis. Commonly used acidic elution methods, low pH levels can dissociate most antibodies-antigen interactions. ■ Multi-mislatoprotte, low purity, face the tricky problem, we first need to analyze the source of miscellaneous proteins: Is it degraded from the protein of interest? Adsorption with filler? Or is it interactive with the protein of interest?

1) Protein partial degradation: use the appropriate protease inhibitor; if it is very sensitive to low pH, it is necessary to add neutralization in advance in the receiving tube before elution. 2) Protein non-specific binding on the antibody, gel or centrifuge wall, it is recommended to prepare the lysate, remove the sample to the new centrifuge tube before the last washing; the washing effect is not Good, it is recommended to increase the number of washing time and the number of times, increase the concentration of NaCl or detergent in the wash buffer.

Fourth level WB

Here to copy a job, 3, 2, 1, and I don't believe this evil! Break Western Blot Metaphysics.

Congratulations, the shackles are successful! MCE protein purification medium full range, zero yuan trial (product web page, click Apply now! You can get a trial package), value-for-money buy, new taste fresh price; "Thanksgiving", true!

Related Products

Affinity column

AffInity Chromatography Column can be matched with a filler for purifying enzymes, antibodies, antigens, recombinant proteins, and the like having different labels.

Glutathione agarose

Glutathione agarose achieves high yield, high purity GST-labeled protein purification at a highly crosslinked 4% agarose gel.

Protein A agarose

MCE Protein A agarose is a substrate with a highly crosslinked 4% agarose gel, and the chemical method is oriented, and the recombinant Protein A can be efficiently coupled, and the animal ascites, serum, cell secretions are separated. Purification IgG, etc.

Protein G agarose

MCE Protein G agarose is a substrate with a highly crosslinked 4% agarose gel, which is directed by chemical methods, and efficiently couples recombinant Protein G, which can be separated from biological fluids such as animal ascites, serum, cell secretion. Purify IgG or its fragment.

Protein L agarose

MCE Protein L AGAROSE is an affinity chromatographic medium containing Kappa light chain antibody for one step.

Anti-Flag affinity gel

MCE Anti-flag affinity GEL is used for immunoprecipitation, protein purification experiments of Flag tag proteins in bacteria and mammalian cell lysates and in vitro expression systems.

Streptaviarin

MCE StrePTavidin Agarose is a highly crosslinked 4% agarose gel as a substrate, covalently coupled to recombinant streptavidin by chemical methods, more combined with more than 30 μg D-Biotin.

Sulfhydryl coupling agarose

MCE HIGH-AFFINITY IODOACETYL AGAROSE covately couples the IODINE ACETIC ACID derivative by chemical method, which can simply, quickly bind to a mercapto-containing protein, polypeptide, and other biocompansion and form a stable mercaptoether bond, the ligand is fixed after fixation. Proactive purification experiment.

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