Information

Help analyzing SDS-Page gel

Help analyzing SDS-Page gel


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

In this experiment, we transformed a truncation of the NFAT protein sequence into a plasmid vector to be expressed in E.Coli as a fusion protein with GST. We also attempted to transform the normal plasmid without NFAT so that we could get GST alone. After growing up these colonies and selecting positive colonies, we lysed the e.coli. We collected some of this lysate for SDS-PAGE. We then put the lysed GST and GST-NFAT solutions in separate columns with glutathione beads. GST binds glutathione. We washed the column and collected some of the wash for SDS-PAGE. Finally, we eluted the GST and GST-NFAT off the column and saved some for SDS-PAGE.

I have attached a picture of a successful version of the SDS-PAGE.

You want to direct your attention to the first lane, the Molecular Weight Marker (MWM). The rest of the lanes should be interpreted in sets of 3. The first of the three is the crude lysate, the second of the three is the first wash, and the last is the eluted protein of interest. The important lanes for my analysis are 8-10 and 11-13.

I will explain what I know from this SDS-PAGE gel so far. I know the weights of the different bands in the molecular weight marker because I was given a copy of the different proteins in the marker. From this information, I was able to confirm that I isolated my protein of interest in lane 13 and isolated GST-alone in lane 10. I also understand the reason why the lysate lanes are darkest, followed by the wash lanes, and then very clean protein of interest lanes. Also, note that the marker and the elution lanes were loaded with 10 microliters while the other lanes were 2 microliters each. However, there are some things that I do not understand from the gel.
(1) Why are the bands so diffuse/low resolution. I imagine it may be overloading the wells, but I am not convinced because I followed a set procedure.
(2) Why is there large amounts of streaking, especially in lanes 5, 8, 9, 10, and 11.
(3) I isolated my protein of interest because it was a fusion protein with GST (or just GST alone), and then eluted the protein off the glutathione beads via excess glutathione wash. Theoretically, the lanes for the elutions should be free of everything except the protein of interest and glutathione then. This mostly holds true for lanes 4 and 9, which should only have GST and glutathione. However, lanes 7 and 13 should only have GST-NFAT and glutathione. The lanes are not as clear however and seem to have stained numerous other proteins. What is going on?
(4) I was told by a professor that the second band in lane 13 (the GST-NFAT lane) was a degredation product. How can this be? The band does not correspond to the weights of NFAT alone or GST alone. Also, if the NFAT-GST was degraded, wouldn't there be two bands of degredation product?
(5) Why do the GST-alone lanes have what looks like doublet-bands? (Lanes 4, 10).
(6) If we eluted the proteins of interest with excess glutathione, which has a very low molecular weight, why doesn't it show up on the gel? Is it possible that it ran off before everything else?
(7) I have a list of the weights of the bands of the molecular marker. Yet, there is an extra band at the bottom that does not show up on my list. Is it possible my list is incomplete, or is there something else happening?

Finally, I have attached a picture of a failed SDS-PAGE gel.

The only thing that I definitively know was a source of error is the fact that my buffer level fell during the run (due to a leak). The results of this gel are accurate (Lanes 2-7 correspond to lanes 8-13 on the above gel, and the results are almost identical) yet the gel turned out horribly. How can the buffer falling cause this result? Or is there something else that may have contributed to this weird pattern? Finally, the lanes are not as distinct in this gel as the first gel, yet the same comb, and thus the same size and spaced wells were used. So why was there so much bleed between lanes/ lack of space between the lanes?

Thanks in advance for any help.


Here are some thoughts based on my own experiences purifying many proteins and running many SDS-PAGE gels:

(1) Resolution--how fast did you run this gel? Often if you run at higher than say 150V or so you can get bands that look like this. The resolution could also be related to…

(2) I would bet the streaking is due to salt in the sample--a lot of the time lysis/wash/elution buffers can have upwards of 500 mM or 1 M salt, and this invariably causes streaking. Load less sample in these lanes and keep the total volume the same to dilute the salt below 250 mM at least. Other things in your buffers could be causing the streaking, but salt is the most common (let us know the composition of your lysis/wash/elution buffers).

(3) Your purification of GST alone looks very good--often your preps will look much more like the lanes you have indicated, which are not entirely clean. This is quite common for a one step purification--often you need 2-3 for certain proteins to completely remove contaminants. There are several possible sources of these bands:

  1. Endogenous proteins: Many bacterial proteins will non-specifically stick to the beads (this is why we wash), but even thorough washing can still leave some contaminants. These proteins also could be bound to your protein of interest and are co-eluted off of the resin.
  2. Truncation products: Depending where your tag is (let's sat it is on the N-terminus), you can sometimes get incomplete translation products--they have the tag, but they are not full length proteins. These will still bind and elute from the beads because they have retained the tag. Ignore this point if you tag is on the C-terminus, as in that case the tag will only be on full-length protein.
  3. Degradation products: This is related to your later questions--often during the purification your protein will be degraded by bacterial proteases. Did you include a variety of protease inhibitors in your lysis buffer? Keeping all of your samples on ice is also important for this reason.

(4) Understand that degradation products do not mean you get only your protein and the fused GST--they can be a variety of truncations due to degradation by proteases.

(5) I would bet this is just due to your running conditions or the salt in your samples. I think this is probably actually your single GST band.

(6) Glutathione will not show up on the gel--it's formula weight is about 300 g/mol if I recall meaning it's about 300 Daltons, so much smaller than anything that you'll retain on the gel. Further, remember than your stain is for proteins--Coomassie blue binds basic residues on proteins.

(7) I'm not sure which band you are referring to, but I think the very bottom band in your marker lane is simply the dye front. It could also be a degradation product of a protein in your ladder.

As far as the second gel goes--it is all due to the buffer level falling during the run. Without buffer covering the top of the gel there is no current flowing. Probably as the leak was occurring you had uneven distribution of the buffer. Strange things can happen when this occurs (unscientific, I know).


The sodium dodecyl sulfate coats the proteins in proportion to their molecular weight and then confers the same negative electrical charge across all proteins in the sample. The rate of migration of a polypeptide in SDS-PAGE is inversely proportional to the logarithm of its molecular weight. This means that the larger the polypeptide, the slower it migrates in a gel. The molecular weight is determined by comparing the migration of protein spots to the migration of standards. Plots of log molecular weight versus the migration distance are reasonably linear.

The proteins separated by SDS-PAGE are often recovered in a procedure that involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing the sodium dodecyl sulfate from the eluted sample, and finally renaturing the protein for subsequent analysis. Proteins that are eluted from gels are used in varied downstream applications successfully, such as protein chemistry, determination of amino acid composition, identification of polypeptides that correspond to specific enzyme activity, and other purposes.

The analysis of protein concentrations is a significant assay in biochemistry research. The Bradford assay is one of the most widely used methods to determine concentrations of protein, relative to a standard. The technique is based on the formation of a complex between proteins in solution and the dye. This assay is commended for overall use, particularly for assessing concentrations of proteins for gel electrophoresis.. The proteins separated by SDS-PAGE are often recovered in a procedure that involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing the sodium dodecyl sulfate from the eluted sample, and finally renaturing the protein for subsequent analysis.

Proteins that are eluted from gels are used in varied downstream applications successfully, such as protein chemistry, determination of amino acid composition, identification of polypeptides that correspond to specific enzyme activity, and other purposes. The analysis of protein concentrations is a significant assay in biochemistry research. The Bradford assay is one of the most widely used method to determine concentrations of protein, relative to a standard. The technique is based on the formation of a complex between proteins in solution and the dye, Brilliant Blue G.

This assay is commended for overall use, particularly for assessing concentrations of proteins for gel electrophoresis. It is based on observations that absorbance maximum for acidic mixtures of Coomassie Brilliant Blue G-250 that do shift from 465 nm up to 595 nm at a time when protein binding occurs. The assay is effective because of the extermination coefficient of the albumin-dye complex solution is usually constant over a range of 10-fold concentration (Westermeier, Naven & Ho?pker, 2008).

The dye reacts mainly with arginine residues but less with histidine, lysine, tryptophan, tyrosine, and phenylalanine residues. Seemingly, this examination is not all that perfect for acidic or basic proteins. However, it is somewhat sensitive to the bovine serum albumin, even more than most proteins, by a factor of two. Gamma globulin (IgG) is the protein standard of preference. The objective of this


Contents

SDS-PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. In addition, SDS (sodium dodecyl sulfate) is used. About 1.4 grams of SDS bind to a gram of protein, Α] Β] Γ] corresponding to one SDS molecule per two amino acids. SDS acts as a surfactant, masking the proteins' intrinsic charge and conferring them very similar charge-to-mass ratios. The intrinsic charges of the proteins are negligible in comparison to the SDS loading, and the positive charges are also greatly reduced in the basic pH range of a separating gel. Upon application of a constant electric field, the protein migrate towards the anode, each with a different speed, depending on its mass. This simple procedure allows precise protein separation by mass.

SDS tends to form spherical micelles in aqueous solutions above a certain concentration called the critical micellar concentration (CMC). Above the critical micellar concentration of 7 to 10 millimolar in solutions, the SDS simultaneously occurs as single molecules (monomer) and as micelles, below the CMC SDS occurs only as monomers in aqueous solutions. At the critical micellar concentration, a micelle consists of about 62 SDS molecules. Δ] However, only SDS monomers bind to proteins via hydrophobic interactions, whereas the SDS micelles are anionic on the outside and do not adsorb any protein. Α] SDS is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of protein structure. Ε] In SDS concentrations above 0.1 millimolar, the unfolding of proteins begins, Α] and above 1 mM, most proteins are denatured. Α] Due to the strong denaturing effect of SDS and the subsequent dissociation of protein complexes, quaternary structures can generally not be determined with SDS. Exceptions are proteins that are stabilised by covalent cross-linking e.g. -S-S- linkages and the SDS-resistant protein complexes, which are stable even in the presence of SDS (the latter, however, only at room temperature). To denature the SDS-resistant complexes a high activation energy is required, which is achieved by heating. SDS resistance is based on a metastability of the protein fold. Although the native, fully folded, SDS-resistant protein does not have sufficient stability in the presence of SDS, the chemical equilibrium of denaturation at room temperature occurs slowly. Stable protein complexes are characterised not only by SDS resistance but also by stability against proteases and an increased biological half-life. Ζ]

Alternatively, polyacrylamide gel electrophoresis can also be performed with the cationic surfactants CTAB in a CTAB-PAGE, Η] ⎖] ⎗] or 16-BAC in a BAC-PAGE. ⎘]


16.6: Micro-report 5- SDS-PAGE and western blot analysis

  • Contributed by Clare M. O&rsquoConnor
  • Associate Professor Emeritus (Biology) at Boston College

In this report, you will describe the results of the SDS-PAGE and western blots that you used to analyze protein expression in your transformed cells under both repressed and induced conditions. Pay special attention to the degree to which these results confirm or contradict the results of the previous transformation/complementation report. The figure should be labeled in such a way that an experienced scientist is able to understand your results from the figure and legend alone. Many experimental details have been relegated to the M&M section. Lanes should be clearly labeled and the molecular weights of the standards should be included. (The example below is from a different class, where students used SDS-PAGE and western blots to analyze overexpression of yeast proteins expressed from pBG1805. Note that, unlike your experiments, a primary antibody to the HA epitope was used to detect proteins on western blots.)

Materials and Methods: Provide information about transformed strains, incubation conditions, preparation of cell extracts, SDS-PAGE gels and western blots. Reference published procedures when possible, noting any modifications. Subheadings may be helpful.

Transformed strains: Include the names of the strains and plasmids that you used to prepare cell extracts.

Extracts: Include information on the media and incubation times used to manipulate protein overexpression from the strains. Reference the manual for the extraction procedure, noting any modifications.

SDS-PAGE gels: Provide details about the % acrylamide of the gels, running time and voltage used for electrophoresis.

Electrophoretic transfer: Include the time and voltage used to transfer proteins from the SDS- PAGE gel to the PVDF membrane. Refer to the manual for other details.

Western blot: Include the antibodies that you used for the blot and the conditions (time, tem- perature) that you used for each of the incubations. Include the time that you used to detect overexpressed proteins with TMB. (This gives a some sense of the abundance of the protein in your extracts.) You do NOT need to include all the wash steps - just reference the manual.

Results and Discussion - Begin by discussing the SDS-PAGE gel. The SDS-PAGE gel provides a snapshot of cellular proteins and a rough comparison of protein concentrations in different extracts. The staining intensity of a band reflects its abundance in the extract.

  • How did the total amount of protein compare between induced and non-induced samples? (What might this indicate about the different carbon sources?)
  • Did you see any changes in individual bands on the gel? Is it possible to detect the fusion protein against the background of other proteins in the extract? Recall that cells have thousands of proteins and that a band may consist of more than one protein species.

The western blot allows you to detect the fusion protein against the background of other cell proteins. Include a table showing predicted and actual sizes of Met or LacZ proteins detected using the western blot technique. The sizes of the proteins are particularly important. (It is probably not possible to get an exact value of the protein sizes, because of the fuzziness of the standards on the western blots. Nonetheless, you should be able to place the proteins within a certain range.) The plasmid-encoded proteins will be larger than the naturally occurring protein because of the epitope tags encoded by the plasmid. Are the observed sizes what you expected?


Purity analysis SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technology commonly used in protein purification and analysis. SDS-PAGE can separate proteins according to the differences in the charge and the different mobility due to different molecular sizes. If the protein sample has been highly purified and contains only one protein, the results would show a single protein band after SDS-PAGE separation. However, when there are multiple proteins in the protein samples, different proteins can be separated into multiple protein bands through SDS-PAGE. Therefore, SDS-PAGE technology provides a direct way to analyze the purity of protein samples. To meet the research needs of analyzing the purity of protein products, MtoZ Biolabs has developed and optimized a SDS-PAGE based workflow, and provides an accurate analytical service for protein/peptide purification study.

Analytical Principles of SDS-PAGE

SDS is a type of anionic surfactant that can break the hydrogen and hydrophobic bonds in proteins. SDS can bind to proteins in a certain proportion to form SDS-protein complexes, covering the intrinsic charges of proteins. Therefore, the migration speed of all kinds of SDS-protein complexes is only determined by the molecular weight of proteins. Different proteins are separated by SDS-PAGE electrophoresis, followed by protein staining and analysis of protein bands.

SDS-PAGE Analytical Workflow

SDS-PAGE Analytical Procedures:

1. Determination of protein concentration
2. Sample preparation: add 2x loading buffer (5% β-ME) and boil sample for 10 minutes
3. Electrophoretic preparation: gel production, installation of the electrophoretic tank, and preparation of electrophoretic buffer
4. Protein samples loading: according to the protein concentration, take an appropriate amount of processed protein samples and add into the pocket of the gel. After loading all samples, add protein standard marker into the first or last pocket of the gel.
5. Electrophoresis: turn on the power supply, adjust the voltage to 100V, and run the gel the constant voltage.
6. Gel staining: At the end of the electrophoretic separation, staining with R250 stain for 1 hours, followed by destaining of protein gel until background become clean and clear.
7. Analysis of purity and molecular weight of protein samples

• Experiment procedures
• Parameters of SDS-PAGE
• Protein purity results
• Bioinformatics analysis


A Butterfly and Biotechnology

Hi, dear readers, friends, it's been a long time, I wrote here. Sorry for that, was a bit busy in lab! And, you can be happy because of the fact that I was busy, as I got lots of experience to share with you! Let us first start with SDS PAGE!

SDS PAGE - Sodium Dodecyl sulphate - Poly Acrylamide Gel Electrophoresis! This SDS PAGE is done for separating proteins based on their molecular weight. It is a widely used technique and it is very useful for having an idea about the expression of your protein of interest.

Principle:
The name SDS PAGE comes from the fact that this method uses SDS for making your protein uniformly negatively charged and of course, the gel is prepared using poly acrylamide. Why to make the protein negatively charged? Because, here our interest is to separate proteins based only on their molecular weight, but not based on charge and all proteins are not negatively charged like DNA (which is separated based on size using Agarose Gel electrophoresis). SDS is an anionic detergent and it makes your protein negatively charged. Hence your protein moves towards the positive electrode. i.e. anode.

Okay, the principle is fine, but, what are all the possible mistakes that one would do, while doing SDS PAGE? Here, I'll explain some possible mistakes, so that it would help beginners. I recommend you to learn the procedure for SDS PAGE before reading this.


BIOLOGY - TPR/NS

A. Transfer of DNA between phage and bacteria did not occur immediately following insertion of the virus.

B. No 32P was identified in the genetic material of infected step 3 bacteria following bacterial lysis and release of phage virions.

C. 35S-containing coat fragments from phage progeny adsorb to bacteria, although the fragments contain no DNA.

- DNA should have been transferred in the experiment bc it is the genetic material. We have to prove wrong that S instead of P was uptaken.

A. there is a mutation in a segment of DNA that binds a promoter.

B. a missense mutation is found in the gene that codes for the repressor.

C. there is a structural problem with a segment of DNA that binds a repressor.

A. has a genome where nearly all material codes for protein.

B. typically utilizes mitosis for cellular division.

C. can perform catabolic reactions to gain energy from macromolecules.

A. travel farther during SDS-PAGE and elute more quickly during size-exclusion chromatography.

B. travel farther during SDS-PAGE and elute more slowly during size-exclusion chromatography.

C. travel a smaller distance during SDS-PAGE and elute more quickly during size-exclusion chromatography.

SDS-PAGE + dithiothreitol: 3 bands

Which prediction about the enzyme structure is most strongly supported by these observations?

A. Enzyme α contains a high ratio of charged to uncharged residues.

B. Enzyme α contains more than one subunit.

C. Enzyme α contains a low ratio of charged to uncharged residues.

B. low blood osmolality and high blood pressure.

C. high blood osmolality and low blood pressure.

Question asks what physiological conditions would lead to the same response as what is observed during alcohol consumption

During FISH, fluorescently labeled probes are hybridized to chromosome clusters.

This is similar to how probes are used in Southern and Northern blots.

The probe must be single-stranded DNA/RNA if it is going to hybridize or bind to chromosomes.

A. 25% normal with blue eyes, 25% normal with brown eyes, 25% blue eyed with a risk of Crohn's disease and 25% brown eyed with a risk of Crohn's disease.

B. 50% normal with blue eyes and 50% brown eyed with a risk of Crohn's disease.

C. 50% blue eyed with a risk of Crohn's disease and 50% normal with brown eyes.

A. Single-crossover events result in one-way displacement of chromosomal content from one chromosome to another, while double-crossover events always reverse this one-way displacement, resulting in chromosomes identical to the pre-crossover chromosomes.

B. Single-crossover events occur during mitosis when a cell splits into two cells, while double-crossover events can only occur during meiosis when a cell splits into four cells.

C. Single-crossover events affect only the ends of chromosome arms, while double-crossover events can affect segments in the middle of chromosome arms.

A. have a higher probability of displaying trisomy 21.

B. have a lower probability of displaying trisomy 21.

C. have a similar probability of displaying trisomy 21.

A. Transfer of DNA between phage and bacteria did not occur immediately following insertion of the virus.

B. No 32P was identified in the genetic material of infected step 3 bacteria following bacterial lysis and release of phage virions.

C. 35S-containing coat fragments from phage progeny adsorb to bacteria, although the fragments contain no DNA.

A. VEGF levels >400 pg/mL are indicative of diabetes-related complications.

B. Elevated VEGF levels cause PDR to progress even after surgical treatment.

C. VEGF levels >400 pg/mL are indicative of PDR.

A. the number of colonies observed only for CRC157 and CRC184 transfected with pC27-53 would decrease.

B. there would be a decrease in the number of colonies in all trials.

C. the number of colonies observed only for CRC169 transfected with pC27-53 and pC27-53X would increase.

A. travel farther during SDS-PAGE and elute more quickly during size-exclusion chromatography.

B. travel farther during SDS-PAGE and elute more slowly during size-exclusion chromatography.

C. travel a smaller distance during SDS-PAGE and elute more quickly during size-exclusion chromatography.

A. The sample size was too small to ensure that the findings were statistically significant.

B. The western blots in Figure 4 were performed at week 52, when the body weight of the Nbea+/+ mice and the Nbea+/- mice had become equivalent.

C. The dwarf phenotype of the Nbea+/- mice failed to manifest consistently over time.

A. VPA treatment significantly increases the level of HERV-W transcription in schizophrenic samples.

B. the presence of either schizophrenia or bipolar disorder does not significantly increase transcription of HERV-W above normal levels.

C. VPA treatment has a significant effect on transcription of ERV-9.

Here, we are looking for the statement that is not supported by the results shown. Figure 2 displays a significant difference between both groups of schizophrenic samples and the healthy control, implying that schizophrenia increases ERV-9 transcription.

A. TEX11 expression is necessary for normal structural development of the testes.

B. TEX11 protein is involved in the post-meiotic stage of mammalian spermatogenesis.


PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size. To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. For this different protein molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or quaternary structure .The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder(marker).


Innovations

Miniaturization

Miniaturization of electrophoretic techniques has been developed for efficiency: maximizing productivity while minimizing area. These innovations enable experiments to run simultaneously, as well as reduce the time and cost required for running these experiments [7] . A feature of electrophoresis miniaturization is incorporation of separation channels, which work to increase channel efficiency.

Polymers, such as polydimethylsiloxane (PDMS) and poly(methyl methacrylate) (PMMA) are used in modern electrophoretic systems as they can be easily manufactured and are more robust than glass. PMMA notably is used in gel electrophoresis because it does not react with polyacrylamide gels. Different material considerations are used in electrophoresis when making the gel. For example, agarose gel is used for larger scale analysis of macromolecules, specifically larger DNA fragments [8] . Polyacrylamide has a higher resolution than agarose gels for analysis of smaller samples, such as single strands of DNA or protein analysis. The pores in PAGE are smaller and allow for greater discrimination and separation of samples, having versatility at the microscale.

Multidimensional Analysis

Multiple assays for a single sample can analyze between 2-4 different characteristics and improve test efficiency [9] . Multidimensional analysis makes use of analyzing different sample properties, such as combining isoelectric focusing and SDS-PAGE [9],[10] . Isoelectric focusing of the sample is run horizontally and separates proteins based on their isoelectric points (approximate pH where samples have a net neutral charge), establishing a ladder of samples based on a pH gradient. Afterwards, SDS-PAGE utilizes the same gel and runs it vertically, further separating and distinguishing proteins of similar isoelectric points by size. Multi-dimensional analysis exploits the different characteristics of samples (charge, size, etc.) order to comprehensively separate and distinguish samples (Figure 7).


Conclusions

The 2D-gel-based proteome analysis has been successfully used to detect and characterize marker proteins that are idiotypic for a specific physiologic or pathologic state of a cell or tissue. However, it is now apparent that the 2DE-MS/MS approach is unsuitable to detect, identify, and quantify every protein in a sample, a task that seems necessary for the comprehensive analysis and eventual mathematical description of biological processes and systems. For this reason, it is necessary to develop novel techniques that allow for much increased starting amounts while permitting large-scale quantitative comparison of protein expression.



Comments:

  1. Zolozuru

    The portal is just excellent, I will recommend it to my friends!

  2. Tojale

    In it something is. Thanks for an explanation, I too consider, that the easier the better...

  3. Scowyrhta

    finally appeared an atom was already waiting

  4. Dassous

    For a long time searched for such answer

  5. Garai

    I think you are not right. I'm sure. We will discuss it. Write in PM, we will talk.

  6. Kaycie

    It should be said - rough mistake.

  7. Derrik

    Incredible sentence, I like it :)



Write a message