Gels - Science topic

Dear Michael Cagney

Indeed, you tried all possible ways and designed excellent experimental setups. Yet, the following suggestions can be helpful:

1. Please check the enzyme units required for per ug DNA and add accordingly.

2. Since you are not getting 750bp band, may be double digestion is not working. You can still get a linear plasmid band in case of single digestion. To ensure, a reaction can be designed with 3 tubes- first one with SbfI only, second one with NcoI only and third one with both enzymes. Tube 1 and 2 can be of smaller reaction volume. If all three tubes show single band, then we can confirm, both enzymes are working.

3. It is recommended to aliquot buffer in small volumes to avoid repeated freezing and thawing.

4. Although it is HF enzyme, you can try a 100uL reaction volume with an overnight reaction time.

Hope it will work.

Kaushik Shandilya Thank you for your detailed answer.

Hello,

After many attempts I managed to solve some of the problems. Right now, Lambda ladder is quite good (although not fully separated probably due to to short swith time), but the sample bands are smeary, tick and distorted. Current electrophoresis conditions were:

Switch time 6-36s

Voltage 6V

Angle 120

Buffer temperature 14C

Run time 22 h

I have tried adding slimmer slices but it didnt improove anything. I would be realy grateful for any suggestions, because im running out of ideas.

Manuele Martinelli I think, Blue dye doesn't interfere much with the antibody binding. I have done western blotting from Blue native gels (non-fixed gel) several times and it worked fine.

Here, the problem is protein precipitation and fixation as Didier Poncet mentioned.

It is most likely the result of impurities like salts, proteins, or other organic components co-precipitating with the RNA during the isolation process that causes RNA pellets from testis samples to become gel-like during the isolation process. These impurities may prevent the RNA pellet from dissolving in water or RNA resuspension buffer, resulting in the production of a gel-like material rather than a transparent RNA solution.

It looks like there is some Coomassie Blue orecipitate on the gel. Is your Coomassie freshly orepared and filtered before staining? If there is cloudiness besides the Coomassie, try destaining with ethanol rather than methanol.

Yes. I routinely do this to check for over-expression. 60ul of culture + 20ul of 4x sample buffer + reducing agent. I heat 5 minutes at 80C then vortex for 30 seconds to break the DNA and reduce viscosity. Load 10-20ul on PAGE. Then detect with coomassie blue

I agree with the gel extraction being a possible issue. Do you use a column kit or a pellet in resin kit? If it's a column, add an extra wash. If it's a resin/slurry kit, add an extra spin and transfer.

You could also try a different ligase and make sure the buffer is fresh (it does expire, especially if it has been frozen/thawed many times or left out).

I like Quick Ligase from New England Biolabs, you can do plasmid prep, digestion, gel, cleanup, ligate and transform all in one reasonable day.

Good luck!

Make sure the genomic DNA is free of proteins, lipids, salts, and RNA contamination, and try to use different amounts of genomic DNA, between 50-200 ng per reaction.

The probes will have to be sterilized along with the medium in the reactor after the sterilization cool the reactor to the required temperature and calibrate both pH and DO probes.

The assay dictates which tube to use. EDTA and sodium citrate containers contain preservatives that interfere with clinical chemistry assays. Red top clotted serum tubes can be used for any assay a yellow top serum gel tube is used. The only difference is the red top tubes don't contain gel to separate the cells from the serum.

There are tube guides published on the internet.

Maria Eduarda Soares da Silva Look at the publication by Dhatterwal, P., Mehrotra, S. & Mehrotra, R. Optimization of PCR conditions for amplifying an AT-rich amino acid transporter promoter sequence with high number of tandem repeats from Arabidopsis thaliana . BMC Res Notes 10, 638 (2017). https://doi.org/10.1186/s13104-017-2982-1

Additionally, with lithium heparin tubes there is noworry about clot formation (normally) and this is a time saver in getting the blood analyzed.

Have you tried loading s whole cell lysate, pre-sonication? Just pellet some cells, resuspend in gel loading buffer, boil and load.

Separating cells from gels can be done using various methods depending on the type of gel and the specific application. Here are some common techniques:

Choose the most appropriate method based on the type of gel, the nature of the cells, and the downstream applications. Always handle cells and gels gently to maintain cell viability and integrity.

It might still be worthwhile to contact biorad customer services to find out if they supplied any subsidiary companies that sell their goods and who might still have stock of this item or even if they have any used ones that they used as demonstartion models that they could let you have. Your company representative can be very useful for this kind of problem

This does not look like leakage but rather that a fiber or something similar is embedded in your agarose gel and/or that you did not properly mix in the gel staining dye before pouring the gel. Make sure that your buffers are not contaminated with anything and that you properly stir in the dye before casting the gel. Also make sure that the running buffer is not contaminated with anything of unknown origin.

The problem was with the Human Brain Whole Tissue Lysate (Adult Whole Normal), novus. When compared with colorectal cell lysate, this difference was obvious. Thank you all for your comments.

In my above question, I mean "after de-staining".

can you suggest some company outside India? I have already talked to v-proteomics.

First, I would quantify the DNA sample before starting the PCR, reduce the total volume of the reaction, and perform a temperature gradient.

I've finally figured out the answer: the problem was in the electrophoresis equipment: the buffer was leaking and therefore the gel wasn't fully covered. I write this down in case other people have a similar problem, to check this I had to keep an eye on the mAmpere of the machinery (lower than 35 mA but higher than 20 mA).

I thank everyone who has been interested.

This may be due to strong interactional forces between the components of material. This also shows that the material is highly viscous and May be in gel form.

In an Electrophoretic Mobility Shift Assay (EMSA), the mobility of RNA-protein complexes (RNP-RNA) and free RNA is affected by various factors including size, charge, and shape. Here's why RNP-RNA might migrate faster than free RNA at higher protein concentrations . the combination of changes in size, charge, shape, and gel interaction due to the formation of RNP-RNA complexes at higher protein concentrations can lead to faster migration of the complexes compared to free RNA during EMSA.

Hey there P. Saravana Kumari! Let's dive into it. To dissolve cellulose and create a transparent film, you P. Saravana Kumari can use an ionic liquid like 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]). It's known for its ability to dissolve cellulose effectively, resulting in a clear solution.

Now, for turning the dissolved polymer into a gel, you P. Saravana Kumari might want to consider using a gelling agent like gelatin or agarose. These substances can form a gel when the solution cools down or undergoes a change in pH.

As for plasticizers, you P. Saravana Kumari could go for something like glycerol or polyethylene glycol (PEG). These are commonly used to increase the flexibility and durability of the cellulose film while maintaining its transparency.

Hope this helps! Let me know if you P. Saravana Kumari need more details or have any other questions.

To purify the protein, filtering the lysate followed by SEC will not give you pure protein. SEC is usually employed at final stages of purification. You can initially enrich your protein of interest by different methods like ion exchange chromatography, reversible precipitation by ammonium sulphate etc. Once the contaminants are reduced to a significant extent, SEC can prove helpful.

Hello,

I agree with Dr. Paul above, this happens due to formation of heteroduplexes. Your original band contains more than one product, with no noticeable difference in mobility, but the slow moving band is a heteroduplex. On cutting the expected band and Re-PCR, all the three combinations are generated again. We observed this and resolved in our study on such observed heterogeneity during ribosomal DNA ITS region amplification in Asiatic Vigna species (see Saini et al., Genet. Res., Camb. (2008), 90, pp. 299–316.). We also proved the differences (indel lengths, 2 bp and above) among clones by doing heteroduplex analysis by mixing different clones, in that study.

If you are interested in getting the amplicons, instead of band purification go for cloning and sequence.

all the best

Hello Mariana Barber

I've heard that LOBSTR can also remove E. coli contaminants during purification. However, in my case, I opted for a different approach. I utilized the Baculovirus expression system, expressing the protein in insect cells, which resulted in a very pure protein.

Kindly let me know if you require any additional information.

Wishing you the best of luck with your purification.

Lubna

Hey there Paolo Tagliafierro!

Exciting project you Paolo Tagliafierro have there for your master's thesis! Creating a phantom in PVA sounds like a fascinating endeavor. It's great that you're considering upgrading to the PVA from Sigma Aldrich for better results.

Regarding your concerns about using a magnetic stirrer with a maximum of 1500 rpm for gel preparation, I understand your apprehension. While a mechanical agitator might be preferable, you Paolo Tagliafierro can still achieve satisfactory results with the magnetic stirrer.

One "safe" method you Paolo Tagliafierro could consider is to control the speed of the magnetic stirrer carefully. Since the high rpm might introduce air bubbles or other issues, try starting at a lower speed and gradually increasing it until you Paolo Tagliafierro reach the desired mixing efficiency. Additionally, you Paolo Tagliafierro could optimize the viscosity of your solution to minimize the risk of issues arising from the stirring process.

Another approach is to use a combination of gentle stirring and manual mixing. After the initial mixing with the magnetic stirrer, you Paolo Tagliafierro can further blend the solution manually to ensure thorough dispersion and minimize any potential problems caused by the limitations of the magnetic stirrer.

Experimentation and careful observation will be key here. By adjusting your technique and closely monitoring the process, you Paolo Tagliafierro can mitigate any challenges associated with using a magnetic stirrer for PVA gel preparation.

Best of luck with your research, and feel free to reach out if you Paolo Tagliafierro have any more questions!

DDT is commonly used during DNA extraction and does not affect DNA stability. However, it may affect the function of enzymes such as DNA polymerase. But since the marker is also affected I don't think DDT has anything with it. I suggest to check the buffer and gel. By the way, the SDS states that the product should be odorless. This suggests that the odor should not be from the kit product!

In Native PAGE (Polyacrylamide Gel Electrophoresis), the pH value of each component plays a crucial role in ensuring the proper separation and migration of proteins based on their native charge and size. maintaining appropriate pH values for each component in Native PAGE is essential for preserving the native structure and charge of proteins, ensuring accurate separation and analysis. Any deviation from the optimal pH range can lead to protein denaturation, aggregation, or altered migration patterns, affecting the reliability and reproducibility of the results.

The study's findings should be taken tentatively. The results are from one research period alone, which only lasted two years (2022, 2023) and only had 76 participants, whose medical conditions were also not stated. There were a number of studies in the past referenced however, performing the same question of plasma as an alternative to serum, to which the results did align. But even so, this transition to using plasma instead of serum has not been normalized for a reason: and that is due to the long standing tradition of serum being used, and a reluctance of abandoning that tradition.

In the same vein, for lithium heparin in particular, the study's findings show consistency and high quality results with plasma.

@Bidwan Sekhar Thanks

Are your initial samples in a buffer that is high in salt? I have seen similar artifacts occur for that reason as well.

Hi Romain,

Could you please share, What approach you used to verify your knockous? I too am getting 3 bands in my knockout Cell line. What could be the reason? Please help.

In the clinical chemistry laboratory, serum is the routinely used specimen. Obtaining the specimen of choice requires blood clotting for 30-60 minutes. In urgent cases, time is of the utmost importance. Hence, the plasma samples are preferable. The journal of Oguzhan Zengi entitled, "The Transition from Gel Separatory Tubes to Lithium Heparin Gel Tubes in the Clinical Laboratory" assesses the viability of Lithium Heparin Gel Tubes in terms of its implications on Turnaround Time (TAT), hemolysis index values, aspiration errors and to assess the possibility of switching to these tubes in Clinical Chemistry tests and Emergency cases. To further establish the viability of Lithium Heparin Gel Tubes for Clinical Chemistry Tests and Immunoassays necessitates retests and further testing.

The journal "Clinical Laboratory Testing in the Emergency Department: A Six-year Analysis" by Beştemir, A. & Bozdereli Berikol, G. (2023) states that the most common clinical laboratory tests in the emergency department were kidney function tests, liver function tests, acute phase reactants, electrolytes, and CBC. In the article "Comparison of Test Results Obtained from Heparin Gel Tubes and Serum Gel Tubes," Ercan, Ş. (2020), the analytes tested for this study are similar to the analytes tested by Oguzhan Zengi but differ in terms of sample source, wherein Ercan's Study utilizes both healthy and hemodialysis patient as sample source to compare the values generated by lithium heparin gel and serum gel tubes. In this study, 28 analytes, or all of the analytes measured in lithium heparin gel tubes, yield similar results with serum gel tubes. To further establish the viability of heparin tubes in clinical chemistry and urgent cases, it is necessary to test for Complete Liver Function Tests. Complete Liver Function tests include Total Bile Acids, a marker for detecting liver functional changes, and Cholinesterase, which detects liver disease and determines poisoning risks. By conducting such, one can further assess the limitations of lithium heparin gel tubes.

Beştemir, A. & Bozdereli Berikol, G. (2023). Clinical laboratory testing in the emergency department: a six-year analysis. Turkish Journal of Biochemistry, 48(5), 467-474. https://doi.org/10.1515/tjb-2022-0250

Ercan, Ş. (2020). Comparison of test results obtained from lithium heparin gel tubes and serum gel tubes. Turkish Journal of Biochemistry, 45(5), 575-586. https://doi.org/10.1515/tjb-2019-0117

The proportion of tubes with aspiration errors in the total number of samples was 0.88%. The proportional reduction in total aspiration errors and related device warnings was statistically significant two months after switching to plasma tubing. The proportion of specimens with aspiration errors to the total number of specimens was 0.44% between April and May 2022 (X 2=41.718, p<0.001).

The factors to be considered in order to avoid discrepancies in values for LDH measurement include variation in tube brands, plasma contamination with platelets, fragmentation of platelets, and fragmentation of erythrocytes. Platelet residues and lysis in plasma, as well as hemolysis and coagulation in serum, can also have an influence on LDH measurement. Overall, the type of sample, preparation process, and tube selection must all be considered in order to obtain an accurate evaluation and interpretation for your LDH measurement.

Lithium heparin gel tubes are now used in the laboratory due to several advantages over other types of blood collection tubes. In order to avoid clotting or thrombin activation, which in turn stops the synthesis of fibrin from fibrinogen, heparin stimulates the inhibition of thrombin and Factor X. It is advised to use lithium heparin since it is the least likely to affect the outcomes of tests for other ions, such as sodium. Transitioning from gel separatory serum tubes to lithium heparin gel tubes can enhance clinical laboratory efficiency and accuracy by reducing processing time, minimizing sample interference, improving sample quality, and expanding the range of tests that can be performed reliably. However, proper training and adaptation to new protocols are crucial to fully realize these benefits.

When assessing the effects of switching from gel separatory serum tubes to lithium heparin gel tubes in the clinical laboratory, the research study considered several important variables – Hemolysis rates, analyte stability, turnaround times, and specimen rejection rates. Hemolysis rates were determined by comparing the frequency of hemolyzed samples before and after the transition; a lower rate denoted better sample quality. Analyte stability was assessed by comparing the changes in analyte concentrations over time in samples collected using the new tube type to the previous tube type. Turnaround times were tracked to see if the change had an impact on how efficiently laboratory workflows operated; any notable deviations from standard suggested possible problems. To determine whether the change had any effect on sample acceptability and laboratory performance, specimen rejection rates were also examined. In a study conducted by Wei et. al (2010), Anti-coagulated blood samples such as lithium heparin gel tubes, are ready to be tested once they are obtained from the patients compared to serum blood samples which needed an ample amount of time to clot completely before being centrifuged. Moreover, interferences regarding coagulation can still be ongoing for serum samples even after centrifugation compared to plasma samples, which can avoid these clotting-related problems.

Reference: Wei, Y., Zhang, C., Yang, X., & Ji, M. (2010). The feasibility of using lithium-heparin plasma from a gel separator tube as a substitute for serum in clinical biochemical tests. Laboratory Medicine, 41(4), 215-219. https://doi.org/10.1309/lmixvai70ks0uwqi

There are previous studies that show the same result to the TAT. Some studies such as the study of Hetu et al. and Ramakers et al. For Hetu et al.it studied Potassium test and concluded a significant decrease in the average time in terms of sample reception and result confirmation. This is similar to Ramakers et al. wherein there is a decreased median TAT with the use of Barricor tubes. Another study is from Badiou et al. that reported the same result using Barricor tubes instead of gel LIH tubes. In this study, the TAT was reduced due to the use of plasma instead of serum that needs to be clotted which is the factor that increases the TAT.

Since lithium heparin interferes with the lactate dehydrogenase (LDH) test, most clinical chemistry and immunoassay assays cannot be performed using lithium heparinized plasma (LIH) tubes. An anticoagulant that is frequently used in blood collection tubes to stop clotting is lithium heparin. It may, however, obstruct several colorimetric and enzymatic tests, such as the LDH test. Red blood cells, the heart, liver, muscles, kidneys, and other tissues all contain the enzyme LDH. It is essential to cellular metabolism because it helps in glycolysis by changing lactate to pyruvate. Increased blood levels of LDH may be a sign of illness or tissue injury. Lithium heparin can cause interference with the enzymatic reaction or the assay's detection technique, which can lead to false increases or decreases in LDH activity. Inaccurate results from this interference may have an impact on patient diagnosis and therapy.

The transition from gel separator serum tubes to gel separator lithium heparinized plasma (LIH) tubes in the clinical laboratory showed favorable outcomes across various parameters according to the study findings. Most notably, the performance of clinical chemistry and immunoassay tests remained largely unaffected, with LIH tubes generally yielding comparable results to serum tubes, albeit with some analytes exhibiting total error values surpassing allowable limits. However, alternative error thresholds were identified, suggesting LIH tubes could be reliably substituted for serum tubes in most tests, except for lactate dehydrogenase (LDH). Additionally, LIH tubes led to improvements in sample quality, as evidenced by reduced aspiration errors and lower hemolysis index values, indicating enhanced sample integrity compared to serum tubes. Moreover, the transition to LIH tubes resulted in decreased turnaround time (TAT), particularly notable in the emergency laboratory setting, suggesting enhanced efficiency in sample processing and analysis. Overall, these findings underscore the potential benefits of LIH tubes in clinical laboratory settings, highlighting their role in optimizing healthcare delivery and resource utilization.

Most tests showed no significant difference between the serum and LIH tubes. For some analytes, total error (TE) values exceeded the total allowable error (TEa) limits. Insulin TE value did not exceed TEa but consumed nearly all its error budget. Plasma tubes could be used instead of serum tubes for most tests, except for lactate dehydrogenase (LDH). Plasma tubes improved sample quality, reduced the incidence of aspiration errors, and decreased TAT in the emergency laboratory.

2 things are worth trying. Wash the comb thoroughly and clean it well. Wash out the loading wells with a pipette or syringe immediately before loading the samples

... for midori Advance for midori direct add it to the sample

Using primers longer than 100 base pairs (bp) for cloning purposes is not a common practice, but it can be necessary for certain applications, such as incorporating large tags, mutagenesis of multiple sites simultaneously, or cloning sequences with high secondary structure. Long primers allow for the introduction of complex modifications and can facilitate the assembly of sequences with precise control over the genetic architecture. However, working with long primers presents unique challenges and considerations.

Design Considerations

Synthesis and Purification

PCR Optimization

Cloning Strategy

Troubleshooting

Conclusion

While using primers longer than 100 bp for cloning is challenging, it is feasible with careful design, high-quality synthesis, and optimization of PCR conditions. These primers offer flexibility for complex cloning projects but require meticulous planning and execution to ensure success. Always verify the final construct sequence to confirm that the intended modifications have been accurately incorporated.

Take a look at this protocol list; it could assist in understanding and solving the problem.

I have seen degradation when the running buffer has been used many times and the gel has been reused and bugs have grown in the systen that chew up dna. Use new buffer for the gel and tank. The 2 bands mentioned may be that the pcr product contains a snp and under the slightly denaturing conditions of the column purification a heteroduplex forms which runs slower (larger) than the homoduplex product which runs at the expected size

As mentioned by the authors of a journal titled “The transition from gel separatory serum to lithium heparin gel tubes in the clinical laboratory” the reason for a negative bias for LDH was not fully understood. However, it is believed that LDH may be released from platelets and other cells into plasma during centrifugation. However, as claimed by Peake, M. J., Pejakovic, M., Alderman, M. J., Penberthy, L. A., and Walmsley, R. N., in a journal article titled “Mechanism of platelet interference with measurement of lactate dehydrogenase activity in plasma,” platelets are said to impede lactate dehydrogenase activity in plasma under conditions of low osmolality. However, their observations challenge these claims, suggesting that what appears as inhibition may actually stem from optical interference by platelets during the reaction.

Their investigation indicates that when platelet lysis is prevented and optical interference is corrected for, both platelet-rich plasma and platelet-poor plasma, as well as serum, exhibit similar levels of lactate dehydrogenase activity. Additionally, it’s worth noting that platelet contamination can lead to unexpected issues when assessing lactate dehydrogenase with centrifugal analyzers. As mentioned in the journal article, the results may vary significantly depending on the volume of diluent added with the sample, potentially causing both higher and lower readings, introducing considerable within-run fluctuations in activity.

Furthermore, platelets can scatter light during the LDH assay, leading to optical interference that affects the accuracy of the measurement. This interference can result in falsely elevated or depressed LDH levels, depending on the extent of platelet contamination and the specific assay method used. In addition, platelets contain LDH enzymes, which can contribute to the overall LDH activity measured in a plasma specimen. If platelets are not adequately removed or lysed during sample processing, their LDH activity may artificially inflate the total LDH measurement, leading to inaccurate results.

Reference: Peake MJ, Pejakovic M, Alderman MJ, Penberthy LA, Walmsley RN. Mechanism of platelet interference with measurement of lactate dehydrogenase activity in plasma. Clin Chem. 1984 Apr;30(4):518-20. PMID: 6705193.

Use two filter sheets on either side of of gel and blot.

We have included EcoRI and HindIII in our primers so PCR product we ligated should carry these 2 sites Tom Masi

It looks to me like either you massively overloaded the lanes with sample, or more likely, you had a lot of salt or something similar that really disrupted the electrophoresis

Gels are not the most accurate of the tools. Since you are saying that you ran a PCR and it was positive—but you could not see the band on the gel—I assume it is a quantitative PCR. In that case, you could try to run the melting curve analysis. If you get a single peak, then this is like having a single band on the gel, even if you cannot see the band on the gel.

Several kits are available for purifying PCR products even without gel-band cutting. The protocol is already in the kits. Fingers crossed.

Allah Rakha Yaseen I'm cheering you on to overcome your difficulties. I suggest you try a higher percentage of SDS-PAGE gels.

It is twice the amount of LAP and lesser crosslinking time. I crosslink it for 45-60s.

Yup, glycerol is great for flash freezing preserving proteins but it does interfere with crystallization. It does do that, though.

I suggest using a tricine-page modification to get a higher resolution for lower MW especially for the below 20kda...

I agree with Péter Gyarmati

Hey there Diana Cherop,

Sounds like you're experiencing some frustration with your TiO2 nanoparticle synthesis, huh? Let me see if I can help you Diana Cherop troubleshoot this.

When you Diana Cherop add a solution of nitric acid and water to a solution of TTIP in isopropanol, the rapid formation of a white precipitate could be due to the hydrolysis of titanium isopropoxide (TTIP) in the presence of water. This reaction forms titanium hydroxide, which precipitates out of solution.

Now, regarding the formation of a clear solution after the initial white precipitate dissolves, followed by the failure to form a gel, there are a few factors we need to consider:

1. **Hydrolysis of TTIP**: The initial white precipitate you Diana Cherop observe is likely titanium hydroxide, formed due to the hydrolysis of TTIP. However, this precipitate can redissolve in acidic conditions, leading to a clear solution.

2. **Gel Formation**: The formation of a gel typically requires the aggregation or cross-linking of nanoparticles. If your solution remains in a liquid state for days without forming a gel, it suggests that the nanoparticles are not aggregating or cross-linking effectively.

Possible reasons for this could include:

- Inadequate concentration of TTIP: Increasing the concentration of TTIP may promote nanoparticle aggregation.

- Insufficient acid concentration: The addition of nitric acid serves to catalyze the hydrolysis and condensation reactions necessary for gel formation. You Diana Cherop may need to adjust the amount of nitric acid added.

- Incorrect solvent ratio: The ratio of TTIP to isopropanol to water could affect the nanoparticle formation. Experimenting with different ratios might improve gel formation.

3. **Stability Issues**: Even if nanoparticles are formed, their stability in solution can affect gel formation. Factors such as pH, solvent choice, and surface chemistry can influence nanoparticle stability.

In summary, to address your issue, consider optimizing the TTIP concentration, adjusting the acid concentration, and experimenting with different solvent ratios. Additionally, ensure proper control of reaction conditions to promote nanoparticle aggregation and gel formation.

Hope this helps you Diana Cherop get back on track with your nanoparticle synthesis! Let me know if you Diana Cherop need further assistance.

It is a difficult question, and I doubt there is a reliable tool for that. You could enter a known protein sequence into various tools (disulfind, etc') and see if they can confirm the formation of a disulfide bond. If a crystalized version of the protein (or a homolog of it) show disulfide bonds, then the answer may be yes. The following page may be of some use:

https://www.researchgate.net/post/Are_there_any_user-friendly_tools_to_predict_disulfide_bonds_from_a_protein_sequence

Alternatively, you could look up evidence of dimerization in the literature - native gels before and after reduction, size estimation via gel filtration columns, and so on.

Hi Julia,

I just want to share my experience. I also had such problem in the past few days. Finally, I found it is the problem of loading buffer. When I changed to new loading buffer, all bands looked great.

Hope this helps you all.

The intrinsic strength of cement gel is an important property that governs the overall strength of cement-based materials. The intrinsic strength refers to the inherent strength of the cement gel, which is the primary binding phase in cement paste. Here are a few methods commonly used to determine the intrinsic strength of cement gel:

It's important to note that the intrinsic strength of cement gel can vary depending on factors such as the water-to-cement ratio, curing conditions, and the presence of supplementary cementitious materials. Additionally, the measurement of intrinsic strength can be challenging due to the heterogeneous nature of cement gel and the difficulty in isolating it from other phases present in cement paste.

Thank you very much.

No problem! A piece of advice I learned a long time ago is your eyes do not lie to you, a spectrophotometer will.

Good Luck!

Michael J. Benedik I have seen RE cut dna get bigger when the enzyme binds to the target dna but has not cut , Then it looks large ( runs slowly) but addition of 0.05%sds denatures the mixture and the dna runs more like its actual linear size

Thank a lot john.

Thank u Ananthi Rajendran i will definitely try this.

Since I do not have a map of the construct I can not tell you which is which.

Hello Prince Dee

For the attached THP-1 cells which have been differentiated, wash the cells gently with pre-cooled PBS, and detach the cells using 0.25% trypsin. Centrifuge the cell suspension for 5 min at 1000×g. Discard the medium and wash the cell pellet three times with pre-cooled PBS. You may add 150-250ul of precooled PBS per 1×10^6 cells to keep the cells suspended. Carry out repeated freeze-thaw cycles by freezing the cell suspension in the freezer and then thawing the cell suspension at room temperature or 37℃ until the cells are fully lysed. This method of lysis will cause the cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Centrifuge the resultant lysed cells for 10 min at 1500×g at 2-8℃. Discard the pellet and collect the supernatant to carry out the assay. Add protease inhibitor such as PMSF at 0.1 - 1mM in the final volume during cell lysis to prevent protein degradation.

Please note: Do not use RIPA lysis buffer or UREA lysis buffer since there is a possibility of causing deviation due to the introduced chemical substances.

No. But you need to perform the total protein assay (Bicinchoninic acid (BCA) assay) to determine the protein concentration of your lysate. The volume of each sample can be normalized to deliver the same amount of total protein for the assay.

Good Luck!

May consider using Permai fluorescence dye.

Hi Abu Faizal

I'm not familiar with the compound you are trying to make, but if the formula you've written is correct this is a trivalent vanadium (V(3+)) salt. I don't see in your procedure anything obvious that would reduce vanadate (V(5+)) to trivalent vanadium. Citrate is unlikely to do it, although it may reduce V(5+) to V(4+).

By the way, the concentrations seem low for gel formation - not impossible, but just an extra incentive to double-check that you got your procedure right.

Best regards,

Emanuel Cooper