SDS-PAGE - Science method

Hi,

Recently did a 2D electrophoresis, using IEF and SDS-Page on mouse muscle protein.

I will attach a picture of our results but they seem somewhat hard to decipher.

Any help would be appreciated.

Many thanks

I have incubated my protein with thrombin, and it has successfully cleaved the tag from my protein. I was thinking that the thrombin should have show in the SDS-PAGE as well, but I didn't see any band for it. I am so curious about the reason, pleaseshare your thoughts with me! =D Thank you!

I am trying to solubalize the recombinant protein from inclusion bodies. The protocal I follwed is as follows,

1. After conferming the expression of recombinant protein in the form of IB through SDS-PAGE, I lysed the Bl21De3 cells in lysis buffer and collected the IB in form of pellet.

2. The pelet was resuspended in ST buffer ( 50mM Tris, 300mM Nacl, 5mM ZnCl2, 10mM beta-mercaptoethanol) supplemented with 10% sarkosyl.

3. Incubated the IB for overnight at 10oC with continuous shaking.

4. Next day, centrufuged the contents at 20000 rpm for 10 min and collected the supernatant. The supernatant was used for SDS-PAGE analysis.

The problem I am facing is, after treating the IB with sarkosyl also, there is no much improvement in protein solubalization. What will be the possible reason for this? Is there any alternative available to increase the IB solubility?

I've had this issue on and off for a while now, and it's really starting to get on my nerves.

For background:

I don't think there is a problem with my gel recipe or running protocol - everyone else in the lab does the same and they usually get good band resolution. 10% Acrylamide resolving gel, plenty of stacking, fresh 10% APS made before casting. I put a layer of butanol on top of the resolving to even it out and remove it after complete polymerization - usually 1 to 1.5 hours. Maybe the alcohol is drying out the top of the resolving??

30 ug protein samples are denatured at 95 C for 5-10 min in fresh loading buffer that I made. Gels are run at 15 mA through the stacking (50-90 V) and 25 mA through the resolving (120- 200+ V). Any suggestions or tips are appreciated. I've attached a picture of a recent membrane (pvdf) with this annoying problem.

We produced mAbs at my lab (classical 1975 hybridoma tech). One of my clones produced one mAb that was promising. When we constructed it, we used subtractive immunization, which consists of immunizing the mice with a non-tumoral cell line, then giving them a chemotherapy agent following 24h and 48h in order to eliminate proliferating B cells (triggered by exposition to non-tumoral cells). Then, we perform, at the same mouse, a series of immunizations with tumoral cells (same tissue-line cell). After purification we did the SDS page gel with the purified mAb as the sample. Strong IgG bands were easily spotted (150kda, 55kda and 22 kda). This mAb, when used in blotting, diluted at 1:1000 gave us the attached figure. First lane is a non-tumoral cell lysate (RWPE-1), second lane is the tumoral cell lysate PC3 (used as immunogen to produce the mAb) and third lane is another tomoral cell lysate, LNCAP. Here is how I am interpreting it: i) at the first lane, as expected, there is only low non-specific binding. At the other lanes, we have a more strong band (which I suppose is the protein my mAb recognize) but also a lot of other bands. I was expecting to see only one band, because my mAb is trully monoclonal. What am I missing here? Am I interpreting this correctly? Are these bands only a result of poor standardization of my blott? Should I do another blott, with both the primary and secondary antibodies more diluted? Thanks a lot!

- In mammals, about 95% of the milk proteins are made up of casein and whey proteins. Bovine milk is a significant source of protein in several parts of the world such as Asia, Africa and Europe.

- The major portion (80%) (Haug, Høstmark & Harstad, 2007) of bovine milk protein is designated by a casein (CN) group encoded by four tightly linked genes: αs1-CN (CSN1S1), β-CN (CSN2), αs2-CN (CSN1S2) and κ-CN (CSN3), located within a 250 kb piece of bovine autosome 6 (Caroli, Chessa & Erhardt, 2009).

- There were differences in milk composition between the breeds (Lôbo et al., 2017). Protein variants have their use in the study of origin and evolution of breeds of goats. These markers have proved to be useful for parentage determination and population analysis (Groselande et al., 1990).

- The negative charges on SDS destroy most of the composite structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric area (Muayad, 2017).

- Protein separation by SDS-PAGE can be applied to evaluation relative molecular mass, to determine the relative abundance of major proteins in a sample, and to define the distribution of proteins among fractions. The purity of protein samples can be estimated and the progress of a fractionation or purification procedure can be followed. Various staining methods can be used to find out rare proteins and to learn something about their biochemical properties (David, 2012).

How’s your experience about SDS?

I have cloned a gene in Suffle T7 express strain using pET28a vector for expression and in vitro studies. After Ni-NTA column purification of this His Tagged protein, I am getting multiple bands in SDS-PAGE gel instead of one.

What could be possible reasons?

Hi..

I have a question ??

Can I measure the concentration of a protein by depending on the size of its band in the SDS-PAGE gel ??

thank you

Hi. I am trying to detect a recombinant arginine-rich 10KD peptide with high positive net charge using SDS-PAGE. on the tris-glycine SDS-PAGE with 15% and 17% resolving gels and 5% stacking gel i did not observe any band related to the peptide. The peptide has a His6 tag. Could any one please guide me on this issue??

I used to prepare 10% tissue homogenate by weighing the tissue in RIPA with protease and phosphatase inhibitor. After homogenization I used to centrifuge it for 20min at 13000rpm in 4 degree. Protein estimation was done by bradford method. I used o load 40ug of protein. After transfer the ponsue stain gives a nice band picture. But developing time I used to not get any band. Some time I used to get bands but some time not. Can any one please help me?

Hi all,

I was wondering whether it's possible to extract proteins from C. elegans only by adding sample buffer (such as

4X Bolt™ LDS Sample Buffer) + reducing reagent, and incubation at 70^oC (or 95^oC) for 10 min? Has anyone ever tried this...?

Thanks!

Hello all!

I am doing SDS-PAGE for my protein of predicted size of 53.5kda bt unexpectedly I am getting bands of around 60kda.. I have done this 3-4 times and Everytime the required band is about 60kda in induced samples.. I have sequenced my cDNA clone also and it is showing right sequence.

suggestion will be highly appreciated..

Thanks

Sometimes when i run my samples, the dye becomes more diffuse and spreads out more when it enters the resolving gel, while other times it is much narrower and darker...Does it indicate anything, regarding the content of protein in the sample?

I have purified a EGFP-His tagged protein which was there in pET15b vector using Ni-NTA column. The concentration obtained was way too high around 5mg/ml. Before the running the SDS-PAGE the sample was boiled for 10 min. But, on SDS-PAGE I have obtained the the molecular weight of 60 kDa instead of 30 kDa which is the molecular weight of EGFP.

I have purified a monomeric EGFP- His protein with a molecular weight of 30 kDa. But, instead of 30 kDa I am getting a thick band of 60 kDa in SDS-PAGE reducing gel. what is the possible reason for that?

I have purified a monoclonal antibody from a hybridoma solution, and when I runn it on an SDS-PAGE gel I get 2 bands for the light chain. I know that an antibody is 150 KDa and when we boil it in the presence of DTT and run it on an SDS-PAGE gel we should get 2 bands, one for the heavy chain at 50 KDa and the other one for the light chain at 25 KDa (An antibody consists of 2 heavy chains and 2 light chains).

Though we can get 2 bands for the heavy chain (as it can get glycosylated), what is confusing me now is that I am getting 2 bands for the light chain (the difference between the bands is not more than 2-3 KDa).

Can the light chain of the antibody be glycosylated? Or is the antibody getting degraded (it should not as I store in NaN3 and I purified it at a couple of months ago).

Can anyone please help me try to find the reason for getting 2 bands for the light chain of the antibody on the gel?

Histone H3 and H4 were connected with 1 or 2 biotin through a short linker. And then they were stained to a dye through a streptavidin.

Just after the biotinylation, the standard SDS-PAGE gel cannot give desired bands at around 20 kDa.

- The band of biotinylized H3 is slightly more clear than the band of H4, but much lighter than the band of Histone without biotinylation.

- In the image of stained gel, stronger signal can be found in the top wells. So I assume it is aggregation of biotinylized hitone.

Any comment is welcomed!!!

i am doing immunoprecipitation with flag pull down experiment. And in control pull down with NRS sentacruz. I have obtained smear type region in between heavy and light chain in both IP as well as control lane on silver staining after SDS-PAGE run. Above heavy chain no prolem is there. Can anyone suggest me how can i clear that region in IgG lane and get a clear bands in the IP lane ?

specifications are:-

20 MIN blocking of sepharose A with BSA.

3 TIMES 5 mins wash etc.

I am facing a problem on loading the aqueous plant extract on SDS PAGE, every thing was done as per the protocol. ON the same the methanol extract got loaded , but the aqueous extract spills out of the well

I'm working on purification of acetyl xylan esterase, I collected active fractions and wanted to confirm it's molecular weight by zymography, I am not getting single bands on SDS-PAGE by silver staining, among all multiple bands I want to confirm AXE band.

I have so many protocols about preparing loading buffer for BN-SDS PAGE and i could not choose the indicated for my experiments.

The protein I want to express is an intracellular protein (non-secreted). I'm using Baculovirus/insect cells expression system. After mani times, I don't find any expression of my protein. I run a small-scale (2 ml) and 50 ml, I see some bands in SDS-PAGE and W.B., but my corresponding protein band is not thick. I checked from the cloning to the transformation; everything is alright.

If anyone can help me to express my non-secreted protein, I will be very thankful.

Thank you.

Hi

I have been struggling with the purification of a GFP-tagged peptide from E.coli. It seems that the peptide degrades and left GFP alone once the cell is lysed.

Looking at the sequence there exist a DG motif which people point to hydrolysis, approx equal to the degradation site. However, since the GFP-peptide is intact in whole cell and becomes GFP after lysis (sonication) within 15-30 min 4 deg celcius, it really seems too fast for a hydrolysis reaction in standard buffer (PBS / Tris pH 7-8). I thus wonder if the boiling in SDS buffer alters the integrity of my construct.

Any idea if I can run my SDS-PAGE in a less hydrolyzing condition?

cheers

I want to express and purify a protein of bacteria (approximately 121 kDa) using pGEX 4T-1, E. coli BL 21 and Glutathione Sepharose 4B. I have tried out with 0.1, 0.5 mM IPTG, at 30 deg C for 4 hrs. However, I do not see any fusion protein expression on the SDS-PAGE gel. Can anyone help me troubleshoot this? I would like to attach my protocol (Screening pGEX recombinants for tagged protein expression) and results of SDS-PAGE gel in the attached file.

Recently, I get streaking bands of the pure protein I study. I phosphorylate the protein with a modified ATP then I "click" a fluorophore. So the samples loaded contain click reagents. I add 4X loading buffer and then I fill the wells. I do not heat the sample otherwise the phosphate falls off. I load 15 uL of sample corresponding to 0.4 ug. I pour my own gels. The pH of the stacking and resolving buffer are fine. I have attached two examples. The gels were made simultaneously but ran on two different days. Would you have any suggestions for stopping this problem? Thank you Adeline

I'm trying to use IAC to purify a protein using an old prepacked NHS column with a potent antibody, that was coupled 2-3 years ago, and getting bad yields (Wide peak over 4 mL at 2 mAU max, protein not visible on a SDS PAGE gel, but shows up on a western blot when probed with the same antibody). I'm wondering if the antibody or column degraded etc. over time, as it was stored in PBS+azide at 4C for two whole years. (We elute with pH2.5 buffer if that makes any difference).

I have the chromatograms from when the column was fresh, and they look relatively good, with a decent area-under-the curve. But this time, it's dropped. Image is attached. I know columns last forever, but I'm just wondering how long a coupled column is good for.

Any good advice is welcomed. Thank you in advance.

If,suppose my interaction partner trying to form complex with my bait protein,is there is a possiblity to see one thick band(Addition of MW of 2 protein),or two seperate bands?If the answer is 2 seperate bands,then,How can i say these proteins are tend to form complexes?

Recently I am trying to purify a protein having molecular weight 57 KDa. During dialysis, I have used 15 mM HEPES-KOH (pH 7.5) for 16 hours and then 50 mM HEPES-KOH (pH 7.5) for 3 hours. I have maintained low temperature (4⁰C) from the beginning of purification to the end of dialysis and I also have used protease inhibitor before cell lysis. Could you please tell me what is(are) the reason of this degradation in lane 3 and how can I overcome this problem.

N. B. In SDS-PAGE, lane 1 is collected binding wash buffer II, lane 2 is collected Elute buffer before dialysis and lane 3 is Elute buffer after dialysis.

The picture demonstrates that each wells is filling by 50 μl of sample enzyme and loading buffer. the numbers in this image represent the amount of sample enzyme; One Well, is loading buffer control (Right side of the image). The question is that the enzyme which mentioned above can't enter to separating gel. Anybody can help me to realize that what should I do to solve this problem?

The light zones on gel exhibit amylase activity and as it is observable that enzyme still located in well and also in the beginning of resolving gel. Please notice that we performed SDS-PAGE on non-reducing conditions.

I will try tricine gels and gradient gels. Is there any other advice?

Thanks a lot

I am using the "ImageJ" software in order to quantify the protein bands on the SDS-PAGE, but I'm not sure it's the best software for this purpose. Does anyone have the experience of using other software to do this? Is there any professional software for SDS-PAGE densitometry?

Initial transfer performed overnight (16hr) at 30V failed because small and mid sized proteins (smaller than 100kDa) transferred right through the membrane (as evident from Ponceau staining of the blot and Coomassie staining of the gel). Transfer buffer used was Bjerrum Schafer-Nielsen buffer (48 mM Tris, 39 mM glycine, pH 9.2, containing 20% methanol) containing 0.1% SDS. Apparatus used is BioRad Mini-Transblot (tank/wet transfer method). The electrophoresed gel, membrane and filter pads were equilibrated in transfer buffer around 30mins prior to transfer.

Hi, i am trying to express my recombinant protein in BL21 or Rosetta E.coli. The vector is pGEX4T2 I. The predicted MW is 51 kDa. However, the actual MW appears to be lower than the expected MW in SDS-PAGE.(The position of my recombinant protein is 37kDa.) Clone in pGEX4T2 was confirmed by sequencing.

Thanks in advance!

Greetings.

I heard about when make buffer include Tris, set pH using concentrated hydrochloric acid(37%). but SDS-PAGE, loading buffer(10X), Tris-glycine, what I heard set pH for 8.3, use only glycine and NO use HCl or NaOH. (set a pH by Tris and glycine, Tris-base is 30g and glycine is 144g, lots of glycine uses)

I heard about, if use concentrated HCl or NaOH, problem occurs when loading protein. Is it due to strong ionization?

in my SDS PAGE gel

Top and middle is good

but

Bottom part always have smear band

Size of my target protein is 17kDa

on the photoshop, i edit the picture, but also it can not cofirm.

My lab is currently searching for a new human/mouse GLI2 antibody. We were using the sc-28674 from Santa Cruz and were quite satisfied with it, but it is no longer produced, so we tried the new sc-271786 from Santa Cruz and the ab26056 from abcam and both did not give satisfying results.

Dear all, I'm currently working with Avibacterium (close family with Haemophillus). I want to compare the expression of an outer membrane protein across different growth phases. There's no MAb available for this protein and producing it in-house is not an option.

A previous work shows that this protein is 600 kDa and whole-protein comparison (via SDS PAGE) with a knock-out mutant for the gene shows no other proteins are of this weight. We don't have an ultracentrifuge to do membrane protein purification/fractionation. So I want to do whole-cell protein extraction and compare the amount of this protein via SDS PAGE and gel-image analysis.

Currently available protocol is to collect the cells, pellet, resuspend in PBS, add SDS PAGE sample buffer, boil, then load onto the gel and run the electrophoresis. Is there any suggestion on how to maximize the protein yield during the extraction, especially because my objective is not merely to detect but to quantify (relatively between various time points)? Do cells from different growth phases have different susceptibility to the lysis buffers (thus affecting the yield)? Thank you in advance.

I am writing a report and I am supposed to determine the purity of the protein sample? Samples were ran on SDS-PAGE and stained with CBB. Using the Rf value and plotting a standard curve, I was able to determine the molecular weight of the protein. However, I am required to determine the purity of the protein. Please any ideas ir suggestions on how to go about this?

I'm running SDS-PAGE using 12% gels, loading 30 ug of total proteins in each well. The protein ladder seems to be resolving fine, however, samples doesn't have many bands. Incidentally, all the bands sizes are less than 75 kDa. Any ideas for troubleshooting?

my protein after i purified it showed one band on sds page. however, it wont form crystals, after gel filtration it shows 2 peaks. Why is this? is it not purified properly? is it due to proteolysis? and if so how come it isnt shown in the sds page?

I´ve had this problem for a while now that my Ponceau staining shows diffused bands and not ladders like it should. Also i’m not getting any results when trying to detect proteins. In SDS-PAGE I used newly made running buffer as well as in transferring, so I doubt it’s beacuse of that.

I’ve noticed that when I make the gels, the TEMED is very yellow. Isn’t this a sign that it contains oxidizing agents? So could that affect? Also when I take the well comb out pieces of gel get to wells even though the gel is polymerized. So in this case I have problems loading my samples to the wells because the gel pieces separate the sample in different levels in the well (I should describe).

Any ideas?

I have just cloned and expressed a thermophilic alkaline serine protease in E.coli BL21(D3). The protease was expressed as a soluble protein (about 60%) with about 30kDa Mol. wt. (confirmed by western blot). The next step for me was to purify the protein using Ni-NTA resin . My problem was to know the best binding, washing and elution buffers with their best concentration that I can use for a one step purification for the enzyme. I have tried the heat treatment but I could not obtain a pure single band on SDS-PAGE. I will like to know the possible protocol to achieve this purification process within the shortest possible notice. Thanks.

Will the protein's phosphorylation state could be affected if the samples are sitting into the 4X SDS-PAGE Sample Loading Buffer for more than a year?

Hello everyone,

I am working on yeast surface display of influenza virus protein. However, I have tried many times but the western blot showed that M2 and HA fusion expressed but in different size as predicted (26kDa/M2 and 78 kDa/HA) compared to yeast and un-induced yeast.

I have tried cMyc antibodies, M2 Abs and HA Abs. The result showed in attached figure.

Would you please give some advice? I really appreciate for your kindly help.

Wish you a nice day.

People suggested that its a chaperone but I tried washing with ATP & Mgcl2 but couldn't get rid of this contaminant. I am attaching SDS PAGE profile of Ni-NTA purified protein please give suggestions how can I get rid of it .

Thank you.

Hi guys,

Which protocol convenient by SDS-PAGE extract protein for yeast ?

Please recommend me some research, article, paper..,

This was a question in my protein science final, the only question I missed. Any suggestions? All my professor will tell me is i should run SDS page as first step. I wrote Cryo-EM, which is apparently wrong.

Hi there!

I'm purifying a recombinant protein and I always get 2 spots in SDS-PAGE in which one spot has two times more mass than the other. Probably it's a dimer.

I would like to try an oxidative sample buffer so I would be able to break possible disulfide bonds between the protein chains.

Do you know a protocol for preparing an oxidative sample buffer?

Thanks!

The pI of my protein is about 7

Hello fellow colleagues and scientists,

we want to determine the purity of our Protein A purified human antibodies via SDS-PAGE. We observe only one band at 150kDa in the non-reduced SDS-PAGE as we would expect it.

In the reduced SDS-PAGE on the other hand we see bands for all the different antibody fragments, which hints to an incomplete reduction of the disulfide bridges between the fragments. The optimal PAGE would only show the 50kDa and 25kDa bands for the heavy and light chains and nothing else.

As 4x sample buffer we use:

glycerol (30%), tris-base (1M, pH = 6,8 with HCl) (25%), SDS (5%), 2-mercaptoethanol (20%), DTT (400mM), bromophenol blue (0,02%).

Which gives a final reducing agent concentration of 5% 2-mercaptoethanol and 100mM DTT.

We heat the samples for 10 Minutes at 90°C before loading them to the gel. The gels were silver-stained. See them attached.

We also tried alkylation with iodoaceteamide and NEM after and during the heating step, which didn't make a difference at all.

We would be thankful for any hints or suggestions to obtain only the two fully reduced bands of the heavy and light chains from the antibody.

Thanks a lot!

I subjected to SDS-PAGE and WB analysis the whole cell extract of E.coli cells to evealuate if a recombinant protein was expressed. Is it necessary to normalize? how? is there a housekeeping?

Thanks

Laura

Hi,

I am going to do Percoll gradient centrifugation for subcellular fractionation. Before I start I would like to know whether I need to adjust the PH of percoll to 7.4. I use percoll from Sigma (PH 8.5-9.5).

And after centrifugation, can I directly use the fractions for SDS PAGE and WB?

Thank You

Anu

Hi all,

I am using the Ni-NTA magnetic agarose bead from Thermo (Pierce™ Ni-NTA Magnetic Agarose Beads) to do the IP, I want to ask in the final elution step, could I just boil the bead with loading buffer for SDS-PAGE instead of elute it? I don't want to reuse the bead and my sample concentration is too low, elution (>200uL) would too dilute.

Should I boil the beads with protein, then use the magnet stand to remove the bead and use the supernatant for SDS-PAGE? will the protein release from the bead like this? Or I could use the whole boiled beads containing protein for SDS-PAGE? Thanks ahead for any answer!

The sample is a mixture of proteins, doesn't contain one purified protein. However there is only one protein band on my membrane (and my previous gels). I keep getting the same result like in the attached image, even when I increased the amount of the protein extracted and ran SDS PAGE on the same day of the extraction to minimize degradation. I'm thinking of using another extraction procedure. I still want to know other opinions. Do you have any other possibilities to explain the strong bands in the middle?

Can anyone share the experience and protocol of SDS-PAGE & Western blot for schirmer test sample?

Sincerely,

Hi. i am working on protein refolding and i am curious if there are protease remaining in the IB. For now, i am including protease inibitor to make sure that there are no proteolysis. To make sure, i ran sds-page(non-reduced) to see if my target protein was cut by protease (after 1hour incubation in RT). The result showed proper size of my target protein. However, still i want to make sure that there are no protease in my sample. Has anyone experience or has an knowledge of this issue? thank you.

I previously tried gelatin zymography using precast 0.1% gelatin gels (Novex) and it worked fine (gave me some 'clearance zones'). I came accross immersing zymography in some papers and wanted to try it, as it seems cheaper and easier. I have used precast SDS-PAGE gel with no substrate (Mini-Protean) and applied the same protocol as previously. The only change was that 0.1% gelatin was added to my developing buffer. After staining with SimplyBlu SafeStain (also used previously) it seems like only a half of gel took up the stain and unfortunately not the half where I was expecting to see proteolytic activity. Any ideas what went wrong?

Regards,

Jadwiga

I ran a 15% SDS-PAGE gel. After the end of the run the lower bands of the marker/ladder were visible clearly. But when the gel was stained with commassie stain, the lower half of the bands in the gel disappeared. 

Later the same reagents and conditions were used to run a gel of 12.5%, that ran and stained properly. 

What could be reason for such observation?

Resolved my protein (envelope glycoprotein ~ 65 kDa) by 12% SDS-PAGE and transferred to nitrocellulose paper for western blotting. Have detected the same protein before, and it worked, but the last few blots have been staining the same protein white. Why may this be so? I have included pictures...

Hello everyone,

I was wondering if anyone has tried using glutathione (or any other natural reducing agents) to reduce proteins for SDS-PAGE analysis in place of beta-mercaptoethanol. Perhaps there are other co-factors essential for the reduction process? Please advise if there are any protocols available.

I have performed a Far UV CD analysis of my purified protein and obtained the Asc file which has all the details of the wavelength and the ellipticity values in degrees. I have run the K2D3 software and found that my protein has alpha helical nature. As far my protein is concerned it is purified by a two step process- ion exchange chromatography and gel filtration chromatography and I have obtained 3 bands in both reducing and non reducing SDS-PAGE. To plot the CD spectra, I would have to use wavelength on X-axis and on the Y-axis should I represent the ellipticity values in mdeg(the values in Asc file itself) or should I determine the Mean residual ellipticity using the formula?

Please the relative references

Hi,

I have trouble in detecting my protein band of interest (approximately 20 kDa) in SDS-PAGE. Protein bands between 35kDa and 11kDa do not appear in the gel.(SDS page image has been attached)

I have tried both 12% and 15% gel concentration but the problem still remain unsolved. what is the problem?Does anyone have experience regarding this issue?

Thank you

Narges

When expressed in Pichia pastoris, the recombinant protein Antigen 5 (~25 kDa) appears in two bands in SDS-PAGE after purification in affinity chromatography. The supperior band have a molecular weight of ~30 kDa. And the lower band have a molecular weight of ~25 kDa. Does someone have any idea of what the additional band might be? Maybe a glycosylation or peptide signal? We are waiting for the results of the sequencing of both bands.

The image bellow contain the purified protein with two bands.

While pouring both the resolving and the stacking gels inside the glass plates, bubbles are produced because

of the sds due to which the wells do not form properly and so I can't load my samples into them.

Also, does the water layer mixes with the resolving gel (while it solidifies inside the plates) when it is put on the top of it??

During SDS-PAGE separation the sample buffer running faster than sample. Can anyone help me to fix this problem?

When performing ChIP-proteomics/ChIP-MS based techniques, if one chooses to do X-ChIP using standard 1% formaldehyde cross-linking is it necessary to reverse the cross-links before performing mass-spec based proteomics techniques?

Many studies tend to use SDS PAGE prior to mass-spec which would lead to cross-link reversal when samples are boiled. Is it possible to send the beads or eluate directly for analysis?

I am ultimately aiming to purify a linear His-tagged peptide of 6 kDa size, which is expected to obtain in low amounts. I have priorly transformed the E. Coli BL21(DE3) cells, then induced with IPTG in various concentrations at various temperatures/hours. Now, I am trying to see if any expression bands are present. (At the SDS-PAGE, I run the lysates of non-transformed cells, transformed-but-not-induced cells, and induced cells.)

At first, I have performed 12,5% SDS-PAGE, and the peptide ran out of the gel. Then, I have tried 16% Tricine-SDS-PAGE, but my gels were not appropriate (too short) to properly carry out the procedure (8 cm x 8 cm, 1.0 mm). Thus, I was not able to separate the bands. Lastly, I have tried 20% SDS-PAGE, and I had perfectly crisp bands above 10 kDa, but there was nothing visible below that.

So, please suggest me a procedure to visualize clear bands at 6 kDa. Else, if I were to perform a Western Blot with an anti-His antibody, could solely this prove that I have produced my peptide?

Our lab is doing nanovaccine project. To label the antigen Bm86 (75KDa) which is the cattle tick antigen, we used Cy5 tag to conjugate with the proteins and then loaded them to silica nanoparticles(SNPs) . After the labeling, we conducted SDS-PAGE and western blot to confirm the binding between SNPs and Bm86. However, not like the results we got from the unlabeled one, we could not get any band on sds gel but had a band for positive which is the unlabeled Bm86, and for western blot we can not only see bands on the 75KDa position but on the top of wells. Therefore, we assume not all labeled proteins ran through the gel, some of them still left on the top like the situation when we add insufficient loading dye.

Hello Everyone,

Recently I engineered a new fusion gene which contained a promoter region fused to a protein which is a hybrid of two proteins (genetically engineered in a certain fashion). It is expressed in bacteria and is a membrane protein.

Before I test the functionality of this engineered protein (for what it was designed to do), I would like to confirm that the protein is being produced normally. In other words, if my functional assays do not work out, at least I know the protein is being produced properly, and the gene of interest is actually present.

There is not easy functional assay to asses this since it is a new protein. I was thinking of perhaps doing a RT-PCR for the fusion gene of interest to see whether it is being produced. Is there any other control tests that should be done to confirm this protein? Would some type of protein extraction and SDS-PAGE be required?

Thanks.

Adam

Hey guys,

I am new to western blot and SDS-PAGE world.

I am running albumin depleted human plasma samples on the 10% SDS gel with a TGS running buffer.

These are the issues I encounter everytime:

1. I don't see any bubbles after 10-12mins of run time.

2. Neither the sample nor the ladder run beyond a certain length.

I have tried changing cassettes and outer tank and made sure that the Anode and Cathode are plugged in proper.

Any help troubleshooting this issue is much appreciated.

Thank you very much!

Hi, My plan is to do a Western blot after running SDS. My research mentor told me to run two gels. I will stain one gel and transfer another gel to a nitrocellulose membrane for WB. She said that is necessary for us to have more data to analyze and make sure we successfully run the gel. Do you think she is right? I think as long as I stain the membrane with Ponceau after transfer, it should be sufficient to tell if I do everything correctly.

Hi ALL,

Anyone working on S. aureus autolysins. I am trying to find out the role antimicrobial peptides in activation and killing of S. aureus.

Any one give me idea how to prepare samples for SDS-PAGE. I generally incubate S. aureus in the presence of peptides and centrifuge 13000 g for 15 min. then take the supernatural and load onto the gel but not did not get any band.

Please share your experience.

I am running a 10% SDS page. Running buffer is tris-glycine-SDS. But the protein ladder started in a bent condition in the stacking, and the vertical bending continues. Voltage in stacking is 100V, then I reduced to 70 to eliminate high voltage scenario. Can anyone suggest any information for uniform bands?

Also, the resolving gel formed uneven layer in previous attempts, but even layer formed with the use of isopropanol.