Antibody & Protein FAQs

IHC is an immunohistochemical experiment, and the sections used generally include IHC-P (paraffin tissue section) and IHC-F (frozen tissue section).

First of all, customers need to estimate the dosage of antibody according to the antibody instructions & the results of pre-experiments. Different samples will have different antibody dilution ratios due to different antigen expression. For example, in the IHC test of an antibody, the pre-test confirmation is calculated according to the dilution ratio of 1: 50. Under normal circumstances, one slice needs to add 100μl of diluted antibody, that is, one slice needs 2μl of antibody, and 20μl can make about 10 slices.

The species mentioned in the antibody instructions can be confirmed, but there is no guarantee that the antibody can be used in unverified species, even if the sequence homology is high. Whether an antibody will recognize proteins in samples from other species involves many factors and we cannot predict. If there is no other choice, you must consider purchasing antibodies for use in unverified species. We recommend that you compare the immunogen sequence with your target protein sequence. The higher the homology, the greater the possibility of success. If the antibody you purchased is for use in unverified species or experimental applications, product replacement or refunds are generally not available.

General antibody instructions will list the types of experiments for which the antibody has been verified to be suitable (such as: WB, IHC, IP, IF, etc.). If the antibody instructions do not mention the application type, it does not mean that the antibody is not suitable. For this type of analysis application, it may be that we have not done the corresponding verification, or the verification sample is not very effective. Generally, if there is no application or the reactivity is not recommended for customers, please try to follow the verified ones listed in the instructions. Type to choose the right antibody for your experiment. If customers want to try it, they can consult technical support in advance or purchase small specifications for corresponding experiments. However, if the expected results are not achieved, product replacement or refunds are generally not available.

The secondary antibody is selected based on the source of the primary antibody and the customer's experimental needs. First look at the species origin and subtype of the primary antibody used in the experiment. Determine the secondary antibody based on the species source and subtype of the primary antibody: if the primary antibody is rabbit-derived IgG, then choose an anti-rabbit IgG secondary antibody, such as Goat Anti-Rabbit IgG; if the primary antibody is mouse-derived IgM, then choose an anti-mouse IgM secondary antibody, such as Goat Anti-Mouse IgM. Marker selection. Common markers include HRP, Biotin, fluorescein, etc. ELISA commonly uses Biotin-labeled secondary antibodies, followed by a streptavidin-labeled HRP for further signal amplification, and TMB substrate for color development; WB mainly selects HRP-labeled secondary antibodies and uses ECL substrate luminescent solution for color development; IHC generally chooses HRP-labeled secondary antibodies. It is recommended to use an immunohistochemistry kit (E-IR-R217 or the latest E-IR-R220/221), and generally use DAB substrate solution for color development; IF can choose secondary antibodies labeled with different fluoresceins according to experimental needs, such as FITC, Cy3, PE, etc. (try to avoid the autofluorescence of cells), and detect the fluorescence through a fluorescence microscope.

The use of positive control is the basis for determining whether the antibody and detection system work normally in an experiment, and it is also a basis for determining whether the protein to be detected exists in the sample to be detected! Especially when the detected sample is uncertain whether the protein to be detected is expressed. Therefore, when there is no positive result in the experiment, it is best to choose the appropriate positive control, and most of the instructions have recommended positive controls; Or you can search according to the SwissProt or Omnigene database in the instructions, these databases often list the tissues in which the protein is highly expressed, and these samples can be used as suitable positive controls.

Because the same protein in different types of cells may have different post-transcriptional splicing bodies and post-translational modifications, such as glycosylation, ubiquitination, etc., the molecular weight is not the only constant. In addition, the protein You can find on the official Uniprot website that the molecular weights of different subtypes of proteins are different. Therefore, there will be a certain difference between the molecular weight found in the literature and the molecular weight of the antibody in the instructions. (You can check some literature references to confirm the molecular weight of the target protein in the target sample).

The repair conditions are generally high temperature and high pressure or microwave heating repair. For example: Antigen retrieval (high pressure method): Add 10 mmol/ml citrate buffer (pH 6.0) sufficient to submerge the slices in the pressure cooker (preparation: dissolve the antigen retrieval solution in the corresponding volume of ddH2O, mix well), and heat When it boils, place the slices on a heat-resistant plastic slicing rack, put them into the pot, cover the lid, fasten the pressure valve, continue heating, set the holding pressure for 4 minutes, open the vent valve to deflate after the time is up, and the pressure returns to zero. Open the lid, take out the inner pot and let it cool to room temperature. After the solution has cooled to room temperature, take out the slices (about 30 minutes). Antigen retrieval (microwave method): Add 10 mmol/ml citrate buffer (pH 6.0) sufficient to submerge the slices in a beaker (preparation: dissolve the antigen retrieval solution in the corresponding volume of ddH2O, mix well), and heat to boiling , place the slices on a heat-resistant plastic slice rack, put them into a beaker, heat over medium-low heat for 30 minutes, take them out, leave them to cool at room temperature, and take out the slices after the solution cools to room temperature (about 30 minutes). For the selection of IHC repair solution, it is recommended to refer to the antibody instruction manual, or refer to the following scheme: for human samples, it is preferred to use EDTA repair solution with pH 9.0 (clinical samples); for rat/mouse samples, it is preferred to use citric acid with pH 6.0 Repair fluid (scientific research sample).

Bovine serum albumin (BSA) is a monomeric protein is the main component in the blood plasma of cows (38g/1000ml), with a molecular weight of 68kD. Isoelectric point 4.8. Bovine serum albumin is widely used in biochemical experiments, such as as a blocking agent in western blot. If the customer wants to use it as a blocking protein for ELISA experiments depends on the customer's experimental system. Different BSAs have significant differences in ELISA experiment blocking, and the client can judge through pre experiments.

Dilute with TBST, BSA concentration 2-5%.

The blocking time is usually 1.5 hours.

This kit provides a full set of reagents for classic WB experiments, including the reagents required from protein extraction to testing of result. It has the advantages of simple operation, high detection sensitivity, clear background, and strong system stability.

Since PVDF membranes have two pore sizes, which are suitable for transferring target proteins of different molecular weights, we have designed two specifications in order to meet different experimental needs. E-IR-R304A (PVDF membrane 0.45 μm pore size) is recommended for the detection of proteins with a molecular weight greater than 20 kDa E-IR-R304B (PVDF membrane 0.22 μm pore size) is recommended for the detection of proteins with a molecular weight less than 20 kDa.

The E-IR-R305 gel kit is not included in E-IR-R304A and E-IR-R304B, and the gel kit needs to be purchased separately;

E-IR-R304A&E-IR-R304B kit contains filter paper, but does not contain sponge pad

The instruments required for WB experiments include ultrasonic cell crusher or a sonicator, water bath, pipette and pipette tip, centrifuge, electrophoresis tank, SDS-PAGE electrophoresis instrument, film transfer instrument, shaker, and WB imaging system instruments.

Both of these are enzyme inhibitors and their function is to prevent protein degradation. PMSF is often used as a protease inhibitor in biochemical and molecular biology experiments. It is an inhibitor of serine proteases and can inhibit the activities of trypsin, chymotrypsin and AChE. Na3VO4 is a phosphatase inhibitor, inhibiting ATPase, alkaline phosphatase and tyrosine phosphatase. The two products in the kit are 100X and should be added to the PIPA lysis buffer at a ratio of 1:100 before use.

After RIPA lysis buffer lyses the cells, the DNA in the nucleus diffuses into the sample relatively completely, forming a sticky liquid. In this case, it can be sonicated after lysis at 35~40% power using ice bath for a total of 1 min, sonicate for 2 s, and keep the interval 2 s to ensure that the cells were fully lysed and to reduce the viscosity of the sample.

Approximately 1 x 106 cells can be lysed. If a well plate is used, it is recommended to add 100-150 μL RIPA lysis buffer to each well of the 6-well plate.

No, the cell lysis buffer for co-immunoprecipitation is a lysis buffer that lyses cells under non-denaturing conditions. Conventional RIPA lysis contains SDS, which will denature the protein and affect the interaction between proteins. It is recommended to use denaturant-free lysis buffer for co-immunoprecipitation.

If you are making a phosphorylated antibody, you must add Na3VO4 inhibitor. If you are making a normal antibody, you can only add PMSF (protease inhibitor).

If the equipment does not have a medium or lower heating option, you can choose a power of about 100-200W to maintain the liquid in a simmring state (that is, just keep the liquid in a gentle bubble state).

This product is valid for at least one year when stored at -20 ℃, and can be stored for about 1-2 weeks at 4 ℃. It is not recommended to store at room temperature and can be stored at -80 ℃. To avoid the negative impact of repeated freeze-thaw cycles on the product's effectiveness, it is recommended to package and store the product as needed after it is completely dissolved upon receipt.

Room temperature storage will have a certain impact on the effectiveness of the product, and the usability of the product can be verified through pre experiments.

Boiling will have a certain impact on the effectiveness of the product, and the usability of the product can be verified through pre experiments.

The loading buffer will have an impact on the electrophoresis solution. If the overflow is small, it can be ignored. If there is a large overflow, it is necessary to replace the electrophoresis solution with fresh one in a timely manner to avoid affecting the subsequent electrophoresis effect. To avoid buffer overflow, it is recommended to dissolve completely in a temperature bath below 37 ℃ before use.

It is not recommended to perform such an operation. Boiling the protein is difficult to completely mix with the buffer, which affects the electrophoresis effect. Mixing the protein with the buffer before boiling is also beneficial for protein denaturation. When in use, the protein is usually thoroughly mixed with the loading buffer before boiling for denaturation.

If a precipitate is found in the sample buffer, it can be dissolved at room temperature or 37 ℃. After the precipitate is completely dissolved, it can continue to be used. When dissolving, do not immerse in a water bath for a long time or boil directly to avoid the deterioration of certain components in the product.

It will not have any impact. SDS in the buffer can bind to proteins and cause the protein SDS complex to carry a large amount of negative charge, completely covering the protein's own charge.

The denatured sample can be diluted with a buffer solution diluted to 1 ×.

If the protein sample contains a relatively high amount of dimers, a reducing agent DTT can be added to help open the dimers.

If the protein band movement rate is uneven, it is recommended to completely dissolve the loading buffer in a bath below 37 ℃ before adding the protein sample. Mix it evenly with the protein sample and boil for 10 minutes to completely denature the protein sample. If the protein band moves slowly, it is necessary to promptly check whether there is any leakage in the electrophoresis chamber.

The precipitation of denatured protein buffer may be related to the ion concentration in the protein sample. Precipitation occurs during temperature changes and can be dissolved by incubating in a water bath below 37 ℃. If there is still a small amount of sediment that cannot be dissolved, centrifuge and take the supernatant for use.

100℃。

This product is suitable for various electrophoresis buffer systems used in denaturing electrophoresis.

Boiling is for protein denaturation. After adding buffer and boiling, the denatured protein has completely denatured and does not need to be boiled again. After complete dissolution, the sample can be spotted.

Extending the electrophoresis time can separate bromophenol blue from the bands, and for small molecular weight proteins, it is recommended to switch to a more suitable concentration separation buffer system.

Perhaps due to incomplete mixing of glycerol in the buffer, it is recommended to dissolve the buffer completely at room temperature or no higher than 37 ℃ before use.

No, this product contains reducing agents such as DTT and cannot be used in non denaturing electrophoresis.

1 x buffer can be used for boiling directly, but it should be noted that the protein loading buffer obtained after this operation cannot be used for BCA concentration measurement.

This product has no specific requirements for protein concentration, it can be diluted proportionally for use.

The sample buffer needs to be thoroughly mixed before use, and the sample and sample buffer also need to be thoroughly mixed. Boiling and denaturing after thorough mixing can avoid such situations.

Bromophenol blue will change from yellow to blue purple with the pH value, which does not affect the normal use of the product. Similar discoloration does not affect subsequent electrophoresis, and the experiment can continue.

Suggest conducting comparative experiments, setting BSA as the positive control, adding buffer solution to the protein sample lysis buffer, mixing well, boiling and denaturing, and observing the band situation by electrophoresis on the sample.

No need, this product contains DTT, which acts similarly to mercaptoethanol as a reducing agent but with lower toxicity.

The pH value of this product is measured at room temperature and 25 ℃.

This product does not contain reducing agents such as DTT and mercaptoethanol.

This product has no significant effect on its effectiveness when left at room temperature (around 25 ℃) for 24 hours and can continue to be used. But it is recommended to immediately pack the goods and store them at -20 ℃, with no more than 5 repeated freeze-thaw cycles.

This product can extract mitochondrial proteins.

When using this product, the removal solution can be restored to room temperature before use; The same membrane can be cleared of antibodies 1-2 times, usually once is recommended.

DAPI can be excited using excitation light at 405 nanometers. Although the optimal excitation wavelength for DAPI is in the ultraviolet region (approximately 350-360 nanometers), it can also effectively excite under blue light at 405 nanometers. Using a 405 nanometer excitation light source can still activate DAPI and generate blue fluorescence for observing and imaging cell nuclei.

This product is only suitable for staining fixed cells. It is not recommended to directly stain live cells or use DAPI staining to determine cell apoptosis. To assess cell apoptosis, it is recommended to use specialized detection kits such as TUNEL, Annexin V-FITC, etc.

This product can be directly diluted to the working solution concentration according to the instructions during use, without the need for additional pH adjustment.

Not recommended for recycling.

This product contains a buffer salt solution, which will not cause significant changes in pH value when diluted from a concentrated solution to a working solution, and there is no need to adjust the pH value again.

The effective pH range of proteinase K is pH 4.0-12.5, and the optimal pH range is pH 7.5-8.0. Please use a buffer solution with the appropriate pH mentioned above, such as 10mM Tris HCl pH 7.4-7.8, to prepare the proteinase K solution according to the experimental requirements. The commonly used working concentration of proteinase K is 10-100ug/ml, and the storage solution concentration is generally 100-200mg/ml. It should be stored at -20 ℃ and diluted to the working concentration before use to avoid prolonged storage.

No need.

Normally, after fixation in 4% paraformaldehyde, tumor tissue slices can be stored at room temperature for weeks to months. For long-term preservation, a better approach is to transfer the fixed tissue samples to a liquid containing buffer such as PBS and store them in a refrigerator at 4 degrees Celsius to ensure their stability and suitability.

Polyformaldehyde (PFA) is a commonly used tissue fixative, typically used to immobilize proteins and cellular structures in biological samples. Although PFA can help maintain cell structure and protein morphology, PFA fixation may cause damage to the cell membrane due to its permeability, which can fix proteins inside and outside the cell. Additionally, the permeability of different cells may not be consistent. The appropriate fixative should be selected based on which aspect of research will be used for the cell membrane.

The occurrence of foam on the cell membrane may be due to prolonged fixation time, and the fixation time can be appropriately shortened to optimize the fixation results.

Polyformaldehyde is prone to volatilization, so it should be stored in a low temperature sealed and dark place.

When using this product to prepare TBST, the final concentration of Tween should be controlled at 0.1%.

This product can be directly used for dyeing without dilution.

If blue precipitate is found in the dye solution before the experiment, filter it with filter paper before use. Whether the dye solution is ineffective can only be judged based on the staining results.

When using Triton X-100 to lyse cells, the concentration is generally 1%; PBS or TBS can be used for dilution.

This product does not contain sodium azide. You can add it yourself if needed.

All are compatible.

It may be difficult to decolorize because impurities in gel are not removed completely. It can be improved by timing the replacement of deionized water, extending the decolorization time and appropriately increasing the decolorization temperature during the decolorization process.

This product can be reused 2-3 times.

By default, the goods are delivered as mentioned in the manual; If you have special needs, it needs to be determined according to the protein situation. In principle, lyophilized powder protein can be sent in liquid form, how liquid protein cannot be sent in ly powder form.

Our protein comes in two forms: liquid and lyophilized powder. Generally speaking, lyophilized powder can be transported by ice bags, reusable blue ice packs, or dry ice; liquids can be transported by reusable blue ice packs or dry ice.